Asynchronous primed PCR

ABSTRACT

An asynchronous thermal cycling protocol for nucleic acid amplification uses two primers with thermal melting temperatures different by about 10 to 30° C. After the higher melting primer has annealed and polymerase mediated extension, the uncopied, single-stranded target sequence may be hybridized and detected by a probe. DNA probes may be cleaved by the exonuclease activity of a polymerase. The probe may be a non-cleaving analog such as PNA. When a probe is labelled with a reporter dye and a quencher selected to undergo energy transfer, e.g. FRET, fluorescence from the reporter dye may be effectively quenched when the probe is unbound. Upon hybridization of the probe to complementary target or upon cleavage while bound to target, the reporter dye is no longer quenched, resulting in a detectable amount of fluorescence. The second, lower-melting primer may be annealed and extended to generate a double-stranded nucleic acid. Amplification may be monitored in real time, including each cycle, or at the end point. The asynchronous PCR thermal cycling protocol can generate a preponderance of the PCR amplicon in single-stranded form by repetition at the end of the protocol of annealing and extension of the higher melting primer.

I. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/875,211, filed Jun. 5, 2001, which claims the benefit under 35 USC §119(e) of Provisional U.S. Application No. 60/209,883, filed Jun. 6,2000, both of which are incorporated herein by reference.

II. FIELD OF THE INVENTION

The invention relates generally to the field of nucleic acidhybridization, and more particularly, to methods of nucleic acidamplification.

III. INTRODUCTION

Nucleic acid amplification assays comprise an important class ofspecific target sequence detection methods in modem biology, withdiverse applications in diagnosis of inherited disease, humanidentification, identification of microorganisms, paternity testing,virology, and DNA sequencing. The polymerase chain reaction (PCR)amplification method allows the production and detection of targetnucleic acid sequences with great sensitivity and specificity. PCRmethods are integral to cloning, analysis of genetic expression, DNAsequencing, genetic mapping, drug discovery, and the like (Gilliland(1990) Proc. Natl. Acad. Sci., 87:2725-2729; Bevan (1992) PCR Methodsand Applications 1:222-228; Green (1991) PCR Methods and Applications,1:77-90; McPherson, M. J., Quirke, P., and Taylor, G. R. in PCR 2: APractical Approach (1995) Oxford University Press, Oxford). Methods fordetecting a PCR product (amplicon) using an oligonucleotide probecapable of hybridizing with the target sequence or amplicon aredescribed in Mullis, U.S. Pat. Nos. 4,683,195 and 4,683,202; EP No.237,362.

In traditional PCR, oligonucleotide primers are annealed to sequences incomplementary target strands that flank a target sequence of interest,and the annealed primers are extended simultaneously to generatedouble-stranded (ds) copies of the target sequence. The primers areextended by a polymerase, preferably a thermal-stable polymerase(McPherson, M. Ed. (1995) PCR 2: A Practical Approach, IRL Press atOxford University Press, Oxford). Traditionally, the sequences of thetwo oligonucleotide primers used in a PCR are designed and selected tohave equal, or similar, Tm values to promote similar annealing andextension efficiencies.

Asymmetric PCR has found use for production of single-stranded copies ofDNA from target sequences (Gyllensten (1988) Proc. Natl. Acad. Sci USA,85:7652; McCabe, P. (1990) “Production of single-stranded DNA byasymmetric PCR” in PCR Protocols: A guide to Methods and Applications,Innis, M. Ed., Academic Press, Inc., San Diego, pp. 76-83). Unequalamounts of the two amplification primers are used, e.g. 1-5 pmoles and50-100 pmoles, respectively for the low- and high-concentration primers.During the first 20-25 cycles, double-stranded DNA is exponentiallygenerated and, when the limiting primer is exhausted, single-strandedDNA accumulates linearly for the remaining 5-10 cycles. A disadvantageis that the PCR must be run under suboptimal conditions, i.e. lowconcentration of one of the primers. Thus the amplification may beinefficient or may be non-reproducible (Hopgood (1992) BioTechniques,13:82; Hunkapiller (1991) Current Opinion in Biotechnology, 2:92). OtherPCR methods that generate single stranded amplicons include enzymaticdigestion of one strand of a double stranded amplicon, multiplexed setsof primer pairs, nested sets of primers, and inverse amplification.However, each method is cumbersome or has limitations (Higuchi (1989)Nucleic Acids Res., 17:5865; Sarkar (1989) Nucleic Acids Res, 16:5197;Stoflet (1988) Science, 239:491; Bevan (1992) PCR Methods andApplications, 1:22; Gyllensten, U. (1989) “Direct sequencing of in vitroamplified DNA” in PCR Technology: Principles and Applications for DNAAmplification, Erlich, H. Ed., Stockton Press, New York, pp. 50-53).

IV. SUMMARY OF THE INVENTION

The present invention relates to methods of nucleic acid amplification,which include novel thermal cycling protocols for nucleic acidamplification. Detection of the progress, i.e. production ofamplification product, may be facilitated and improved by hybridizing adetectable probe to a single-stranded form of the target sequence. Thesingle-stranded target is an intermediate in the two stage annealing andextension protocol. A first, higher melting primer is selectivelyannealed to one strand of the target and extended, resulting in adouble-stranded copy and the uncopied, single-stranded target.

In a first aspect, the invention includes a method for producingcomplementary polynucleotide strands of a target polynucleotide. Amixture is obtained comprising first and second target polynucleotidestrands which are capable of hybridizing with each other to form abase-paired structure that contains a target sequence, a first primerthat is complementary to a first region in the first targetpolynucleotide strand, and a second primer that is complementary to asecond region in the second target polynucleotide strand, such that thefirst and second regions flank the target sequence. The first primer isextended at a first temperature to form a first complex comprising afirst complementary strand that is hybridized to the first targetstrand, under conditions such that the second primer does notsubstantially hybridize to the second region. The second primer isextended at a second temperature that is lower than the firsttemperature, to form a second complex comprising a second complementarystrand that is hybridized to the second target strand. Before extendingthe second primer, a detectable probe is hybridized to a complementarybinding region in the second target strand, and the hybridized probe isdetected as a measure of second target strand.

In another aspect, an asynchronous thermal cycling protocol comprisesthe steps of:

annealing a first primer to a first strand of a denatured target nucleicacid at a first annealing temperature;

extending the first primer with primer extension reagents at anextension temperature or the first annealing temperature to generate adouble-stranded nucleic acid, wherein the primer extension reagentscomprise a polymerase, nucleotide 5′-triphosphates, and a buffer;

annealing a detectable probe to a second strand of the denatured targetnucleic acid at a probe hybridization temperature;

annealing a second primer to the second strand of the denatured targetnucleic acid at a second annealing temperature wherein the secondannealing temperature is lower than the first annealing temperature andextension temperature;

extending the second primer with primer extension reagents at theextension temperature to generate a double-stranded nucleic acid; and

denaturing the double-stranded target nucleic acid into a first strandand a second strand at a denaturing temperature.

By the above method of the invention, a detectable probe is annealed tothe uncopied, single-stranded target. This hybridization event isdetected, e.g. by FRET when the probe has a reporter/quencher pair oflabels. The probe may be DNA and cleaved by nuclease activity of thepolymerase. Alternatively, the probe may be non-cleavable. The probe maybe a nucleic acid analog or chimera comprising nucleic acid analogmonomer units, such as 2-aminoethylglycine. The probe may be PNA or aPNA/DNA chimera. PNA FRET probes may be comprised of a sequence of2-aminoethylglycine with nucleobase units, flanked by a reporter andquencher pair.

The probe may be detected while hybridized to target. Detection of theprobe may be conducted each cycle during a PCR (real-time).Alternatively, probe may be detected or quantitated at the end of PCR,e.g. after completion of 2 to 50 cycles, or more, of geometric or linearamplification (end-point).

After probe detection, a second primer with a lower Tm than the firstprimer is selectively annealed to the single-stranded target andextended to make a copy of the target. The asynchronous thermal cyclingmethod with probe detection can be repeated through a number of cycleswhere the mixture undergoes temperature changes to effect the steps ofdenaturation, annealing, and primer extension at defined temperaturesfor defined timed periods.

During one embodiment of an asynchronous thermal cycling protocol, aprobe specifically hybridizes to the amplified nucleic acid. Whenhybridized, the nuclease activity of the polymerase may degrade theprobe by internucleotide cleavage, thereby eliminating theintramolecular quenching maintained by the intact probe. Because theprobe is designed to hybridize specifically to the amplified targetnucleic acid (amplicon), the increase in fluorescence intensity from thePCR reaction mixture, caused by cleavage of the probe, can be correlatedwith the progress of amplification, i.e. the amount of target sequenceand amount of amplification.

In general, the target nucleic acid in the sample will be a sequence ofDNA, most usually genomic DNA. However, the present invention can alsobe practiced with other nucleic acids, such as a syntheticoligonucleotide, messenger RNA, ribosomal RNA, viral RNA, cDNA, orcloned DNA. Suitable target nucleic acid samples include single ordouble-stranded DNA or RNA for use in the present invention.

In another aspect, the invention includes a method for producingcomplementary polynucleotide strands of a target polynucleotide. Amixture is obtained comprising a first and second target polynucleotideswhich are capable of hybridizing with each other to form a base-pairedstructure that contains a target sequence, a first primer that iscomplementary to a first region in the first target polynucleotide, anda second primer that is complementary to a second region in the secondtarget polynucleotide, such that the first and second regions flank thetarget sequence. The first primer is extended at a first temperature toform a first complex comprising a first complementary strand that ishybridized to the first target strand, under conditions such that thesecond primer does not substantially hybridize to the second region. Thesecond primer is extended at a second temperature that is lower than thefirst temperature, to form a second complex comprising a secondcomplementary strand that is hybridized to the second target strand. Thefirst and second complexes may be denatured. The steps of first primerextension, second primer extension, and denaturation steps may berepeated in one or more cycles.

In another aspect, the invention includes an asynchronous thermalcycling method for producing an excess of ss amplicon, comprising stepsof:

annealing a first primer to a first strand of a denatured target nucleicacid at a first annealing temperature;

extending the first primer with primer extension reagents at anextension temperature or the first annealing temperature to generate adouble-stranded nucleic acid, wherein the primer extension reagentscomprise a polymerase, nucleotide 5′-triphosphates, and a buffer;

annealing a second primer to a second strand of the denatured targetnucleic acid at a second annealing temperature wherein the secondannealing temperature is lower than the first annealing temperature andextension temperature;

extending the second primer with primer extension reagents at theextension temperature to generate a double-stranded nucleic acid; and

denaturing the double-stranded target into a first strand and a secondstrand at a denaturing temperature.

The cycle of steps can be repeated for 2 to 50 cycles or more to producedouble stranded (ds) amplicon. The steps of annealing the second primerand extending the second primer can be omitted in the last 1 or morecycles to produce an excess of single-stranded (ss) amplicon.

In another aspect, the invention includes a method of characterizingcDNA libraries by sequence determination, viz. sequencing byhybridization (SBH).

In another embodiment, this invention is related to kits suitable forperforming a PCR assay by an asynchronous thermal cycling protocol whichdetects the presence or absence of a target sequence in a sample nucleicacid. The kits may allow real-time or end-point detection orquantitation of one or more target sequences in a sample. In oneembodiment, the kits comprise primers with melting point differences ofabout 10 to 30° C. The kits may also include one or more probes,nucleotides, polymerase, and other reagents or compositions which areselected to perform the PCR, or measure and detect a target.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic for an asynchronous PCR thermal cycling methodaccording to one embodiment of the present invention, including stepsof: (i) denaturing double stranded target, (ii) annealing a firstprimer, (iii) extension of the first primer, (iv) probe hybridization,(v) annealing a second primer, and (vi) extension of the second primer.The temperatures and times are exemplary.

FIG. 2 shows a schematic for hybridization of a first primer (longarrow) at a higher temperature than a second primer (short arrow) to atarget nucleic acid according to one embodiment of the presentinvention.

FIG. 3 shows a schematic for hybridizing a primer and probe todouble-stranded (partially) target during traditional PCR (top) andhybridizing a primer and FRET probe (F=reporter dye, Q=quencher) tosingle-stranded target during asynchronous PCR by a probe (bottom)according to one embodiment of the present invention.

FIG. 4 a shows asynchronous PCR (top), according to one embodiment ofthe present invention, and traditional PCR (bottom) thermal cyclingprotocols, with sequential and cyclical duration at specifictemperatures.

FIG. 4 b shows polyacrylamide (15%) gel electrophoresis analysis underdenaturing conditions (about 55-60° C., 7M urea) and SYBR-Green stainingof amplicons after three PCR protocols: asynchronous, traditional, andasymmetric (top), and a schematic of amplification of target DNA withthree combinations of forward and reverse primers (bottom). Forwardprimers are 5′ labelled with an electrophoretic mobility modifier, e.g.biotin or FAM.

FIG. 5 shows an exemplary PNA FRET probe including a reporter dye (F)and a quencher (Q) with glutamic acid and lysine linkages (top). Theprobe exists in at least one conformation when unhybridized to acomplementary target which causes quenching of the reporter dye (bottomleft). Upon hybridization to target, quenching is diminished andfluorescence intensity increases (bottom right).

FIG. 6 shows fluorescence intensity measurements over time on the ABI7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.)of a 16 nt PNA FRET probe (SEQ ID NO:1): without complementary DNA(top); hybridized to the duplex form of complementary 68 nt DNA (SEQ IDNO:2) and 74 nt DNA (SEQ ID NO:3) (middle); and hybridized tocomplementary 68 nt ss DNA (SEQ ID NO:2) (bottom).

FIG. 7 a shows the change in fluorescence (ΔRn) measured on the ABI 7700during asynchronous PCR when a 14 nt PNA FRET probe (SEQ ID NO:8)hybridizes to its perfect match, single G-T mismatch, and single C-Tmismatch complementary targets.

FIG. 7 b shows the change in fluorescence (ΔRn) measured on the ABI 7700during asynchronous PCR when a 16 nt PNA FRET probe (SEQ ID NO:1)hybridizes to its perfect match, single G-T mismatch, and single C-Tmismatch complementary targets.

FIG. 8 shows the change in fluorescence (ΔRn) measured on the ABI 7700during asynchronous PCR with an 8 nt PNA FRET probe (SEQ ID NO:11) and a9 nt PNA FRET probe (SEQ ID NO:12) amplified by an asynchronous thermalcycling protocol according to one embodiment of the present invention,at the bottom.

FIG. 9 shows the two-fold increase in fluorescence intensity from a 16nt PNA FRET probe (SEQ ID NO:1) during the course of an exemplary,averaged asynchronous PCR thermal cycle and an averaged traditional PCRthermal cycle. Each plot is averaged from cycles 25-30 of 40 totalcycles. The temperature profile is shown.

FIG. 10 shows the change in fluorescence (ΔRn) measuring real-timequantification using PNA probes on the ABI 7700 during PCR when 15 nt(SEQ ID NO:14), 16 nt (SEQ ID NO:1), and 17 nt (SEQ ID NO:15) PNA FRETprobes each detect 6 samples: 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, and 10⁹ startingcopies of 68 nt synthetic ss DNA target during an asynchronous PCRthermal cycling protocol according to one embodiment of the presentinvention.

FIG. 11 shows the linear correlation between a threshold cycle ofdetectable geometric amplification (C_(T)) and starting copy number of68 nt synthetic ss DNA target from FIG. 10 during an asynchronous PCRthermal cycling protocol according to one embodiment of the presentinvention.

FIG. 12 shows the display of data from the ABI 7700 for real-timequantification of PCR using a traditional PCR thermal cycling protocolon with a 16 nt PNA FRET probe (SEQ ID NO:1), 10⁴ to 10⁹ starting copiesof 68 nt synthetic ss DNA target, and the same other reagents as in FIG.10.

FIG. 13 shows a schematic for an asynchronous PCR thermal cyclingprotocol, according to one embodiment of the present invention, with lowtemperature hybridization temperature (30-37° C.) of low Tm, short PNAFRET probes.

FIG. 14 a shows a schematic example of the first two cycles of a PCRthermal cycling protocol with a 5′ (GC)₄ clamp primer, followed by anasynchronous thermal cycling protocol.

FIG. 14 b shows the change in fluorescence (ΔRn) measured on the ABI7700 during PCR when 16 nt PNA FRET probe (SEQ ID NO:16) hybridizes toits perfect match complementary target in the K-ras gene during anasynchronous PCR thermal cycling protocol with: (A) equal Tm primers,(B) a 5′ (GC)₄ clamp primer, and (C) disparate Tm primers.

FIG. 15 a (top) shows the change in fluorescence (ΔRn) measured on theABI 7700 during the traditional PCR thermal cycling protocol with equalTm primers and eight target samples containing amounts of β-actingenomic target ds DNA differing by increments of 5 (left to right:50,000, 10,000, 2000, 400, 80, 16, 3, 0.6 pg. Amplicon was detected bythe nuclease cleavage method with a DNA FRET probe (SEQ ID NO:23).

FIG. 15 b (bottom) shows the change in fluorescence (ΔRn) measured onthe ABI 7700 during an asynchronous PCR thermal cycling protocol withdisparate Tm primers and the eight target samples from 0.6 to 50,000 pg(right to left) of β-actin genomic target ds DNA of FIG. 15 a. Ampliconwas detected by a nuclease cleavage assay with a DNA FRET probe (SEQ IDNO:23).

FIG. 15 c shows a schematic for PCR detection by nuclease cleavage of aDNA FRET probe using primers of equal Tm and the traditional PCR thermalcycling protocol (top) and exemplary primers of disparate Tm and anexemplary asynchronous PCR thermal cycling protocol (bottom).

FIG. 16 shows the thermal cycling protocols for the traditional PCRthermal cycling in FIG. 15 a and the exemplary asynchronous PCR thermalcycling protocol employed in FIG. 15 b.

FIG. 17 shows homogeneous detection of PCR cDNA clones with PNA FRETprobes by sequencing-by-hybridization (SBH).

FIG. 18 shows a schematic for a method of PCR including exponentialamplification with two disparate Tm primers by an exemplary asynchronousthermal cycling protocol, followed by a number of cycles of ahigh-temperature protocol where hybridization and extension areconducted at a temperature high enough such that only the higher Tmprimer anneals and extends.

FIG. 19 shows an experimental design and comparison of a traditional PCRprotocol and an exemplary asynchronous thermal cycling protocol withdetection and quantitation of ss and ds amplicons by denaturingpolyacrylamide gel electrophoresis (PAGE).

FIG. 20 a shows polyacrylamide (15%) gel electrophoresis analysis underdenaturing conditions (about 55-60° C., 7M urea) and SYBR-Green stainingof the products from asynchronous PCR. The resulting ss DNA separatedfrom duplex are quantitated by densitometry and expressed as a ratio ofthe upper to lower bands in each lane.

FIG. 20 b shows the asynchronous PCR thermal cycling protocol for theexperiment of FIG. 20 a.

FIG. 21 shows the structure of an exemplary FAM and DABCYL labelled PNAFRET probe structure: 6-FAM-Glu-NH-PNA-C(O)-Lys-Lys-DABCYL, where n isthe number of 2-aminoethylglycine units.

FIG. 22 shows array fluorescent signal image results of a comparison ofhybridization of 5′ labelled PCR products, generated by a traditionalthermal cycling protocol (left) and an asynchronous thermal cyclingprotocol (right) according to one embodiment of the present invention.

VI. DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with theexemplary embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover all alternatives, modifications, andequivalents, which may be included within the scope of the claimedinvention.

VI.1 Definitions

“Nucleobase” means a nitrogen-containing heterocyclic moiety capable offorming Watson-Crick hydrogen bonds in pairing with a complementarynucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or apyrimidine. Typical nucleobases are the naturally occurring nucleobasesadenine, guanine, cytosine, uracil, thymine, and analogs of thenaturally occurring nucleobases, e.g. 7-deazaadenine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine, nebularine,nitropyrrole, nitroindole, 2-amino-purine, 2,6-diamino-purine,hypoxanthine, pseudouridine, pseudocytidine, pseudoisocytidine,5-propynyl-cytidine, isocytidine, isoguanine, 7-deaza-quanine,2-thio-pyrimidine, 6-thio-guanine, 4-thio-thymine, 4-thio-uracil,O⁶-methyl-guanine, N⁶-methyl-adenine, O⁴-methyl-thymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, andethenoadenine (Fasman (1989) Practical Handbook of Biochemistry andMolecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla.).

“Nucleoside” refers to a compound consisting of a nucleobase linked tothe C-1′ carbon of a ribose sugar. The ribose may be substituted orunsubstituted. Substituted ribose sugars include, but are not limitedto, those riboses in which one or more of the carbon atoms, for examplethe 2′-carbon atom, is substituted with one or more of the same ordifferent Cl, F, —R, —OR, —NR₂ or halogen groups, where each R isindependently H, C₁-C₆ alkyl or C₅-C₁₄ aryl. Ribose examples includeribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g. 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(WO 98/22489; WO 98/39352; WO 99/14226). LNA sugar analogs within anoligonucleotide are represented by the structures:

where B is any nucleobase.

Modifications at the 2′- or 3′-position of ribose include hydrogen,hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloroand bromo. Nucleosides and nucleotides include the natural D opticalisomer, as well as the L optical isomer forms (Garbesi (1993) Nucl.Acids Res. 21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435;Urata, (1993) Nucleic Acids Symposium Ser. No. 29:69-70). When thenucleobase is purine, e.g. A or G, the ribose sugar is attached to theN⁹-position of the nucleobase. When the nucleobase is pyrimidine, e.g.C, T or U, the pentose sugar is attached to the N¹-position of thenucleobase (Kornberg and Baker, (1992) DNA Replication, 2^(nd) Ed.,Freeman, San Francisco, Calif.).

“Nucleotide” refers to a phosphate ester of a nucleoside, as a monomerunit or within a nucleic acid. Nucleotides are sometimes denoted as“NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. “Nucleotide 5′-triphosphate” refers to anucleotide with a triphosphate ester group at the 5′ position. Thetriphosphate ester group may include sulfur substitutions for thevarious oxygens, e.g. α-thio-nucleotide 5′-triphosphates.

As used herein, the terms “polynucleotide” or “oligonucleotide” are usedinterchangeably and mean single-stranded and double-stranded polymers ofnucleotide monomers, including 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺ and the like. A polynucleotidemay be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof. Polynucleotides may becomprised of nucleobase and sugar analogs. Polynucleotides typicallyrange in size from a few monomeric units, e.g. 5-40 when they arefrequently referred to in the art as oligonucleotides, to severalthousands of monomeric nucleotide units. Unless denoted otherwise,whenever a polynucleotide sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, unless otherwise noted.

“Internucleotide analog” means a phosphate ester analog or anon-phosphate analog of an oligonucleotide. Phosphate ester analogsinclude: (i) (C₁-C₄) alkylphosphonate, e.g. methylphosphonate; (ii)phosphoramidate; (iii) (C₁-C₆) alkyl- or substitutedalkyl-phosphotriester; (iv) phosphorothioate; and (v)phosphorodithioate. Non-phosphate analogs include wherein thesugar/phosphate moieties are replaced by an amide linkage, such as a2-aminoethylglycine unit, commonly referred to as PNA (Nielsen (1991)Science 254:1497-1500).

“Attachment site” refers to a site on a moiety or a molecule, e.g. adye, an oligonucleotide, or a PNA, to which is covalently attached, orcapable of being covalently attached, a linker.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches a one moiety or molecule, e.g. adye to a polynucleotide, or one dye to another.

“Reactive linking group” refers to a chemically reactive substituent ormoiety, e.g. a nucleophile or electrophile, capable of reacting withanother molecule to form a covalent bond.

“Heterocycle” refers to a molecule with a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur (asopposed to carbon).

“Enzymatically extendable” refers to a nucleotide which is: (i) capableof being enzymatically incorporated onto the terminus of apolynucleotide chain through the action of a polymerase enzyme, and (ii)capable of supporting further primer extension. Enzymatically extendablenucleotides include nucleotide 5′-triphosphates, i.e. dNTP and NTP.

“Enzymatically incorporatable” refers to a nucleotide which is capableof being enzymatically incorporated onto the terminus of apolynucleotide chain through the action of a polymerase enzyme.Enzymatically incorporatable nucleotides include dNTP, NTP, and2′,3′-dideoxy, nucleotide 5′-triphosphates, i.e. ddNTP.

“Target sequence” means a polynucleotide sequence that is the subject ofhybridization with a complementary polynucleotide, e.g. a primer orprobe. The target sequence can be composed of DNA, RNA, an analogthereof, and including combinations thereof.

The term “probe” means an oligonucleotide that forms a duplex structureby complementary base pairing with a sequence of a target nucleic acid.In the present invention, probes may be labelled, e.g. with afluorescent dye, or a pair of labels comprised of a fluorescent reporterdye and quencher, to enable detection.

The term “label” refers to any moiety which can be attached to amolecule and: (i) provides a detectable signal; (ii) interacts with asecond label to modify the detectable signal provided by the secondlabel, e.g. FRET; (iii) stabilizes hybridization, i.e. duplex formation;or (iv) provides a capture moiety, i.e. affinity, antibody/antigen,ionic complexation. Labelling can be accomplished using any one of alarge number of known techniques employing known labels, linkages,linking groups, reagents, reaction conditions, and analysis andpurification methods. Labels include light-emitting compounds whichgenerate a detectable signal by fluorescence, chemiluminescence, orbioluminescence (Kricka, L. in Nonisotopic DNA Probe Techniques (1992),Academic Press, San Diego, pp. 3-28). Another class of labels arehybridization-stabilizing moieties which serve to enhance, stabilize, orinfluence hybridization of duplexes, e.g. intercalators, minor-groovebinders, and cross-linking functional groups (Blackburn, G. and Gait, M.Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology,2^(nd) Edition, (1996) Oxford University Press, pp. 15-81). Yet anotherclass of labels effect the separation or immobilization of a molecule byspecific or non-specific capture, for example biotin, digoxigenin, andother haptens (Andrus, A. “Chemical methods for 5′ non-isotopiclabelling of PCR probes and primers” (1995) in PCR 2: A PracticalApproach, Oxford University Press, Oxford, pp. 39-54).

The term “quenching” refers to a decrease in fluorescence of a firstmoiety (reporter dye) caused by a second moiety (quencher) regardless ofthe mechanism.

“Chimera” as used herein refers to an oligonucleotide including one ormore nucleotide and one or more nucleotide analog units.

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in formation of a duplex or otherhigher-ordered structure. The primary interaction is base specific, i.e.A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.

The term “end-point analysis” refers to a method where data collectionoccurs only when a reaction is substantially complete.

The term “real-time analysis” refers to periodic monitoring during PCR.Certain systems such as the ABI 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) conduct monitoring during each thermalcycle at a pre-determined or user-defined point. Real-time analysis ofPCR with FRET probes measures fluorescent dye signal changes fromcycle-to-cycle, preferably minus any internal control signals.

VI.2a Synthesis of Primers and Probes

Oligonucleotides are commonly synthesized on solid supports by thephosphoramidite method (Caruthers, U.S. Pat. No. 4,973,679; Beaucage(1992) Tetrahedron 48:2223-2311), using commercially availablephosphoramidite nucleosides (Caruthers, U.S. Pat. No. 4,415,732),supports, e.g. silica, controlled-pore-glass (Caruthers, U.S. Pat. No.4,458,066) and polystyrene (Andrus, U.S. Pat. Nos. 5,047,524 and5,262,530) and automated synthesizers (Caruthers, U.S. Pat. No.4,458,066; Models 392, 394, 3948, 3900 DNA/RNA Synthesizers, AppliedBiosystems, Foster City, Calif.).

VI.2b Primer and Probe Design and Selection

PCR primers and probes to practice the asynchronous thermal cyclingprotocols and for comparative experiments with the traditional andasymmetric thermal cycling protocols may be designed using PrimerExpress™ (Version 1.0, Applied Biosystems, CA). Other oligonucleotideselection and evaluation software programs have been reported or arecommercially available. A target nucleic acid sequence is entered orimported from a database, e.g. genetic code such as GenBank(http://www.ncbi.nlm.nih.gov/; Nuc. Acids Res. 2000 Jan. 1;28(1):15-8).In some embodiments, the binding site location of primers complementaryto a target are selected to amplify amplicons of a particular length ata particular site. In other embodiments, the binding site of a primermay be unknown, as in the use of universal primers, i.e. a set ofrandom-priming primers, or primers with redundant-base or promiscuousbase-pairing nucleotides.

Upon heating, a duplex melts and undergoes a hyperchromic shift. The Tmfor a particular primer or probe is that temperature at which half thepopulation is hybridized to target. The Tm is noted as an inflectionpoint in the characteristic sinusoidal curve which results from plottingthe absorbance, e.g. at 260 nm, versus temperature. Hybridizationaffinity is affected by primer length, G+C content, salt concentration,chemical modifications of the primers, e.g. 2′-O-methyl (Stump (1999)Nucleic Acids Res. 27:4642-48), labels on the primers, and reagentswhich may stabilize, e.g. intercalators, or destabilize, i.e.denaturants, duplex formation. Tm values of the primers and probes maybe designed by selection of some combination of parameters includingsequence, length, G+C content, and hybridization stabilizingmodifications to have particular Tm values to effect efficientamplification in a particular asynchronous thermal cycling protocol.

The sequence and length of primers used in the asynchronous PCR methodsare selected such that annealing to target of a first, higher-meltingprimer occurs at a first annealing temperature where a second,lower-melting primer does not anneal to the target. A pair, or set ofpairs, of primers are selected to establish an approximate 10 to 30° C.difference in the Tm between the higher-melting and lower-meltingprimer. As an example, FIG. 2 shows a higher-melting primer of a pairmay be designed to have a Tm of about 60-75° C. and the lower-meltingprimer may be selected to have a Tm of about 45-55° C. The Tm values maybe estimated using standard base-pairing and nearest-neighboralgorithms. Typically, annealing of primers and probes to target isconducted at temperatures at, or up to 10° C. below, the estimatedmelting temperature of the duplex (Ausubel, et al Eds. “Preparation andAnalysis of DNA”, and “The Polymerase Chain Reaction” in CurrentProtocols in Molecular Biology, (1993) John Wiley & Sons, New York.

The Tm value for a probe may be 68-70° C., except shorter high-affinityprobes, e.g. PNA FRET probes, which may have a lower Tm. Probe sequencesare selected to be complementary to the target polynucleotide and inbetween the primer binding sites of the target. The probe sequenceshould be selected to be complementary to the strand which is extendedby the second, lower Tm primer. This strand will be substantiallysingle-stranded after annealing and extension of the first, higher Tmprimer to copy the other strand (FIG. 1). Probe sequences may bedesigned to include non-target specific, self-complementary sequencesthat favor enforced proximity of a reporter dye label and a quencherlabel. The self-complementary sequences may be located at the 5′ and 3′termini of the probe. Such “hairpin” sequences have an intramolecular“stem” region and a non base-paired “loop” region. Upon binding totarget, the reporter dye and quencher are spatially separated andfluorescence increases.

Probes are designed to be not extendable by polymerase during PCR. PNAFRET probes are generally not substrates for polymerase. DNA probes maybe rendered non-extendable by blocking the 3′ termini with a 3′phosphate or other group at the 3′ hydroxyl or nucleobase of the 3′terminal nucleotide (Livak, U.S. Pat. No. 5,723,591).

VI.2c Nucleic Acid Analogs

Nucleic acid analogs are structural analogs of DNA and RNA and which aredesigned to hybridize to complementary nucleic acid sequences. Throughmodification of the internucleotide linkage, the sugar, and/or thenucleobase, nucleic acid analogs of the invention may attain any or allof the following desired properties: 1) optimized hybridizationspecificity or affinity, 2) nuclease resistance, 3) chemical stability,4) solubility, 5) membrane-permeability, and 6) ease or low costs ofsynthesis and purification.

One useful and accessible class of nucleic acid analogs is the family ofpeptide nucleic acids (PNA) in which the sugar/phosphate backbone of DNAor RNA has been replaced with acyclic, achiral, and neutral polyamidelinkages. The 2-aminoethylglycine polyamide linkage with nucleobasesattached to the linkage through an amide bond has been well-studied asan embodiment of PNA and shown to possess exceptional hybridizationspecificity and affinity (Buchardt, WO 92/20702; Nielsen (1991) Science254:1497-1500; Egholm (1993) Nature, 365:566-68).

VI.2d PNA Fret Probes

PNA can hybridize to its target complement in either a parallel oranti-parallel orientation. However, the anti-parallel duplex (where thecarboxyl terminus of PNA is aligned with the 5′ terminus of DNA, and theamino terminus of PNA is aligned with the 3′ terminus of DNA) istypically more stable (Egholm (1993) Nature 365:566-68). PNA probes areknown to bind to target DNA sequences with high specificity and affinity(Coull, U.S. Pat. No. 6,110,676). The PNA FRET probe examples of thepresent invention, with reporter and quencher moieties, are designedsuch that the PNA anneals in the anti-parallel orientation with thetarget sequences. Whereas PNA probes bound to complementary targetsequences are generally not appreciably cleaved by nuclease activity ofa polymerase during PCR, hybridization alone may cause sufficientseparation of the reporter and quencher moieties to result in anincrease in fluorescence by a decrease in quenching (FIG. 5).

PNA may be synthesized at any scale. Most conveniently, PNA issynthesized at the 2 μmole scale, using Fmoc/Bhoc, tBoc/Z, or MMTprotecting group monomers on an Expedite Synthesizer (AppliedBiosystems) on XAL or PAL support; or on the Model 433A Synthesizer(Applied Biosystems) with MBHA support; or on other automatedsynthesizers. The PNA FRET probes may be synthesized on many of thesolid supports commonly used for peptide synthesis. For reviews ofsolid-phase peptide synthesis, see: J. Stewart and J. Young, “SolidPhase Peptide Synthesis”, Pierce Chemical Co. Rockford, Ill., 1984; E.Atherton and R. C. Sheppard, “Solid phase peptide synthesis: A practicalapproach”, IRL Press, Oxford, 1989; M. W. Pennington and B. M. Dunn(Eds.) “Methods in molecular biology, Vol. 35: Peptide synthesisprotocols”, Humana Press, Totowa, N.J. (1994), pp. 91; G. Grant (Ed.),“Synthetic peptides”, W.H. Freeman & Co., New York, N.Y., 1992; G. B.Fields, Int. J. Peptide Protein Res. (1990) 35:161; A. J. Smith in“techniques in protein chemistry III”, R. Angeletti (Ed.), AcademicPress, Orlando, Fla., 1992, pp. 219; G. B. Fields (Eds.), “Methods inenzymology: Vol. 289”, Academic Press, New York, N.Y., 1997; W. C. Chanand P. D. White, “Fmoc solid phase peptide synthesis: a practicalapproach, Oxford University Press, Oxford, UK, 2000; P. Lloyd-Williamsand F. Albericio (Eds.), “Chemical approaches to the synthesis ofpeptides and proteins”, CRC Press, New York, N.Y. 1997.

After synthesis is complete, the crude PNA may be cleaved from thesupport, e.g. with trifluoroacetic acid, and then precipitated withdiethylether and washed twice in diethylether. PNA may be purified byreverse-phase HPLC, analyzed by mass spectroscopy, and quantitated bycorrelating absorbance at 260 nm with mass. Fluorescent-labelled PNAprobes have demonstrated desirable properties in hybridization assays(Hyldig-Nielsen, U.S. Pat. No. 5,985,563; Coull, WO 98/24933; Coull, WO99/22018; Gildea, WO 99/21881; Coull, WO 99/49293).

PNA-DNA chimera are oligomer molecules with discrete PNA and nucleotidemoieties. They can be synthesized by covalently linking PNA monomers andnucleotides in virtually any combination or sequence. Efficient andautomated methods have been developed for synthesizing PNA-DNA chimera(Vinayak (1997) Nucleosides & Nucleotides 16:1653-56; Uhlmann (1996)Angew. Chem., Intl. Ed. Eng. 35:2632-35; Uhlmann, EP 829542; Van derLaan (1997) Tetrahedron Lett. 38:2249-52; Van der Laan (1998) Bioorg.Med. Chem. Lett. 8:663-68. PNA-DNA chimera are designed to havedesirable properties found in PNA and DNA, e.g. superior hybridizationproperties of PNA and biological functions like DNA, including primerextension through the 3′ OH terminus of the DNA moiety (Uhlmann (1998)Biol. Chem. 379:1045-52).

The linker between the PNA monomer units and labels include: (i) acovalent bond; (ii) an alkyldiyl spacer —(CH₂)_(n)—, where n is 1 to 12;(iii) ethyleneoxy —(CH₂CH₂O)_(n)—, where n is 1 to 12, (iv) aryldiyl(C₆-C₂₀); or (v) one or more amino acids. Lysine, aspartic acid, andglutamic acid side chains may be linkage sites in PNA FRET probes. Theε-amino group of the sidechain of lysine may be the reactive linkinggroup for attachment of a label, e.g. reporter dye or quencher. Linkersare typically attached to the amino and/or carboxyl terminus of the PNAby the corresponding monomer units with compatible protecting groups andreactive functionality for condensation with PNA monomer units and thesolid support. For example, the “O linker”, units of2-(2-aminoethoxy)acetic acid, can be attached to the amino terminus ofany PNA backbone amino group, or on amino functionality of a solidsupport.

VI.2e Labelling

Labelled oligonucleotides may be formed by reacting an appropriatereactive label and an oligonucleotide in a suitable solvent in whichboth are soluble, using methods well-known in the art, for example, seeHermanson, Bioconjugate Techniques, (1996) Academic Press, San Diego,Calif. pp. 40-55, 643-71. The crude, labelled oligonucleotides may bepurified from any starting materials or unwanted by-products, and storeddry or in solution for later use, preferably at low temperature.

The label may bear a reactive linking group at one of the substituentpositions, e.g. 5- or 6-carboxy of fluorescein or rhodamine, forcovalent attachment to an oligonucleotide or nucleotide through alinkage. Generally, the linkage linking a label and the oligonucleotideor nucleotide should not (i) interfere with primer extension, (ii)inhibit polymerase activity, or (iii) adversely affect the fluorescenceproperties of a dye label, e.g. quenching or bleaching. Reactive linkinggroups are moieties capable of forming a covalent bond, typicallyelectrophilic functional groups capable of reacting with nucleophilicgroups on an oligonucleotide such as amines and thiols. Examples ofreactive linking groups include active esters, e.g., isothiocyanate,sulfonyl chloride, sulfonate ester, silyl halide, 2,6-dichlorotriazinyl,phosphoramidite, maleimide, haloacetyl, epoxide, alkylhalide, allylhalide, aldehyde, ketone, acylazide, anhydride, and iodoacetamide.Active esters include succinimidyl (NHS), hydroxybenzotriazolyl (HOBt)and pentafluorophenyl esters.

One reactive linking group of a fluorescent dye is anN-hydroxysuccinimidyl ester (NHS) of a carboxyl group substituent of thefluorescent dye. The NHS ester of the dye may be preformed, isolated,purified, and/or characterized, or it may be formed in situ and reactedwith a nucleophilic group of an oligonucleotide. Typically, a carboxylform of the dye is activated by reacting with some combination of: (1) acarbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate); (2) an activator, such as 1-hydroxybenzotriazole(HOBt); and (3) N-hydroxysuccinimide to give the NHS ester of the dye.

Another reactive linking group of a label is a phosphoramidite form offluorescent dyes, quenchers, minor groove binders, and mobilitymodifiers. Phosphoramidite dye reagents are particularly useful for theautomated synthesis of labelled oligonucleotides. The phosphoramiditereagents can be nucleosidic or non-nucleosidic. Non-nucleosidic forms ofphosphoramidite dye reagents having the general formula:

effect labelling of an oligonucleotide with a single fluorescent dye.DYE is a protected or unprotected fluorescent dye. Alternatively,instead of a fluorescent dye, DYE may be a quencher, a minor groovebinder, or a mobility modifier. L is a linker. R²⁴ and R²⁵ takenseparately are C₁-C₁₂ alkyl, C₄-C₁₀ aryl, and cycloalkyl containing upto 10 carbon atoms, or R²⁴ and R²⁵ taken together with thephosphoramidite nitrogen atom form a saturated nitrogen heterocycle. R²⁶is a phosphite ester protecting group which prevents unwanted extensionof the oligonucleotide. Generally, R²⁶ is stable to oligonucleotidesynthesis conditions yet is able to be removed from a syntheticoligonucleotide product with a reagent that does not adversely affectthe integrity of the oligonucleotide or the dye. R²⁶ may be: (i) methyl,(ii) 2-cyanoethyl; —CH₂CH₂CN, or (iii) 2-(4-nitrophenyl)ethyl;—CH₂CH₂(p-NO₂Ph).

The general phosphoramidite dye reagent above reacts with a hydroxylgroup, e.g. 5′ terminal OH of an oligonucleotide bound to a solidsupport, under mild acid activation, to form an internucleotidephosphite group which is then oxidized to an internucleotide phosphategroup. In some instances, the dye may contain functional groups thatrequire protection either during the synthesis of the phosphoramiditereagent or during its subsequent use to label molecules such asoligonucleotides. The protecting group(s) used will depend upon thenature of the functional groups, and will be apparent to those havingskill in the art (Greene, T. and Wuts, P. Protective Groups in OrganicSynthesis, 2nd Ed., John Wiley & Sons, New York, 1991). The dye will beattached at the 5′ terminus of the oligonucleotide, as a consequence ofthe 3′ to 5′ direction of synthesis. Other phosphoramidite dye reagents,nucleosidic and non-nucleosidic allow for labelling at other sites of anoligonucleotide, e.g. 3′ terminus, nucleobase, internucleotide linkage,sugar. Labelling at the nucleobase, internucleotide linkage, and sugarsites allows for internal and multiple labelling with fluorescent dyes.

Nucleotide 5′-triphosphates may be labelled for use in certainembodiments of the invention. The sugar or nucleobase moieties of thenucleotides may be labelled. Nucleobase labelling sites include the 8-Cof a purine nucleobase, the 7-C or 8-C of a 7-deazapurine nucleobase,and the 5-position of a pyrimidine nucleobase. The labelled nucleotideis enzymatically incorporatable and enzymatically extendable. Labellednucleotide 5′-triphosphates have the following formula:

where DYE is a protected or unprotected dye, including energy transferdye. Alternatively, DYE may be a quencher, biotin, a minor groovebinder, or a mobility modifier. B is a nucleobase, e.g. uracil, thymine,cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine. R¹⁹ istriphosphate, thiophosphate, or phosphate ester analog. R²⁰ and R²¹,when taken alone, are each independently H, HO, and F. Linker L mayinclude alkynyl, propargyl, propargylethoxyamido, vinyl, and allylgroups. For example, L may be:

wherein n is 0, 1, or 2 (Khan, U.S. Pat. Nos. 5,770,716 and 5,821,356;Hobbs, U.S. Pat. No. 5,151,507).

A nucleobase-labelled oligonucleotide primer or probe may have thefollowing formula:

where the primer or probe comprises 2 to 100 nucleotides. DYE is afluorescent dye, including energy transfer dye. B is a nucleobase, e.g.uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and8-deazaguanosine. L is a linker, e.g. propargyl, propargylethoxyamido,allyl, vinyl, or C₁-C₁₂ alkyldiyl. R²¹ is H, OH, halide, azide, amine,C₁-C₆ aminoalkyl, C₁-C₆ alkyl, allyl, C₁-C₆ alkoxy, —OCH₃, or—OCH₂CH═CH₂. R²² is H, phosphate, internucleotide phosphodiester, orinternucleotide analog. R²³ is H, phosphate, internucleotidephosphodiester, or internucleotide analog. In this embodiment, thenucleobase-labelled oligonucleotide may bear multiple fluorescentlabels, e.g. dyes, attached through the nucleobases. Nucleobase-labelledoligonucleotides may be formed by: (i) enzymatic incorporation ofenzymatically incorporatable nucleotide reagents where R¹⁹ istriphosphate, by a DNA polymerase or ligase, and (ii) coupling of anucleoside phosphoramidite reagent by automated synthesis (Theisen(1992) “Fluorescent dye phosphoramidite labelling of oligonucleotides”,in Nucleic Acid Symposium Series No. 27, Oxford University Press,Oxford, pp. 99-100). Whereas, nucleobase-labelled oligonucleotides maybe multiply labelled by incorporation of more than one incorporatablenucleotide, labelling with a phosphoramidite dye label reagent leads tosingly 5′-labelled oligonucleotides, according to the following formula:

where X is O, NH, or S; R²¹ is H, OH, halide, azide, amine, C₁-C₆aminoalkyl, C₁-C₆ alkyl, allyl, C₁-C₆ alkoxy, —OCH₃, or —OCH₂CH═CH₂; R²²is H, phosphate, internucleotide phosphodiester, or internucleotideanalog; and R²³ is H, phosphate, internucleotide phosphodiester, orinternucleotide analog. L is a linker, including C₁-C₁₂ alkyldiyl, e.g.n-hexyldiyl, aryldiyl, or polyethyleneoxy (U.S. Pat. No. 4,757,141;Andrus, “Chemical methods for 5′ non-isotopic labelling of PCR probesand primers” (1995) in PCR 2: A Practical Approach, Oxford UniversityPress, Oxford, pp. 39-54; Hermanson, Bioconjugate Techniques, (1996)Academic Press, San Diego, Calif. pp. 40-55, 643-71; Mullah (1998) Nucl.Acids Res. 26:1026-1031.

A variety of labels may be covalently attached at the 3′ terminus ofoligonucleotide probes. A solid support bearing a label, or bearingfunctionality which can be labelled by a post-synthesis reaction, can beutilized as a solid support for oligonucleotide synthesis (U.S. Pat.Nos. 5,141,813; 5,231,191, 5,401,837; 5,736,626). By this approach, thelabel or the functionality is present during synthesis of theoligonucleotide. During cleavage and deprotection, the label or thefunctionality remains covalently attached to the oligonucleotide.Oligonucleotide probes labelled at the 3′ terminus may have thefollowing formula:

where the probe comprises 2 to 100 nucleotides. DYE may be a fluorescentdye, a quencher, a minor groove binder or other label. DYE may be acombination of labels, such as a minor groove binder and a quencher. Bis a nucleobase, e.g. uracil, thymine, cytosine, adenine,7-deazaadenine, guanine, and 8-deazaguanosine. L is a linker, e.g.propargyl, propargylethoxyamido, allyl, vinyl, or C₁-C₁₂ alkyldiyl. R²¹is H, OH, halide, azide, amine, C₁-C₆ aminoalkyl, C₁-C₆ alkyl, allyl,C₁-C₆ alkoxy, —OCH₃, or —OCH₂CH═CH₂. R²³ is internucleotidephosphodiester or internucleotide analog.

In one post-synthesis chemical labelling method an oligonucleotide islabelled as follows: An NHS form of 6-carboxy fluorescein is dissolvedor suspended in DMSO and added in excess (10-20×) to a 5′-aminohexyloligonucleotide in 0.25 M bicarbonate/carbonate buffer at about pH 9 andallowed to react for 6 hours (Fung, U.S. Pat. No. 4,757,141). The dyelabelled oligonucleotide product can be separated from unreacted dye bypassage through a size-exclusion chromatography column eluting withbuffer, e.g., 0.1 molar triethylamine acetate (TEAA). The fractioncontaining the crude labelled oligonucleotide can be further purified byreverse phase HPLC employing gradient elution.

Oligonucleotide primers and probes of the present invention may belabelled with moieties that affect the rate of electrophoreticmigration, i.e. mobility-modifying labels. Mobility-modifying labelsinclude, but are not limited to biotin, fluorescent dyes, cholesterol,and polyethyleneoxy units, —CH₂CH₂O)_(n)— where n may be 1 to 100(Grossman, U.S. Pat. No. 5,624,800). Preferably, n is from 2 to 20. Thepolyethyleneoxy units may be interspersed with phosphate groups.Specifically labelling fluorescent-labelled primers with additionallabels of polyethyleneoxy of discrete and known size allows forseparation by electrophoresis of amplicons, substantially independent ofthe size, i.e. number of nucleotides, of the amplicon. That is,polynucleotides of the same length may be discriminated by detection ofspectrally resolvable dye labels and separated on the basis ofmobility-modifying labels. Polynucleotides bearing both dye labels andmobility-modifying labels may be formed enzymatically by ligation orpolymerase extension, e.g. asynchronous PCR, of the single-labelledoligonucleotide or nucleotide constituents.

One class of labels provides signals for detection of labelled extensionand amplification products by fluorescence, chemiluminescence, andelectrochemical luminescence (Kricka, L. in Nonisotopic DNA ProbeTechniques (1992), Academic Press, San Diego, pp. 3-28).Chemiluminescent labels include 1,2-dioxetane compounds (U.S. Pat. No.4,931,223; Bronstein (1994) Anal. Biochemistry 219:169-81). Fluorescentdyes useful for labelling probes, primers, and nucleotide5′-triphosphates include fluoresceins, rhodamines (U.S. Pat. Nos.5,366,860; 5,936,087; 6,051,719), cyanines (Kubista, WO 97/45539), andmetal porphyrin complexes (Stanton, WO 88/04777).

Fluorescent reporter dyes include xanthene compounds such asfluoresceins I and rhodamines II:

The ring positions of I and II may be substituted. The amino R groups ofII may be substituted. The substituents include covalent attachments tothe primers, probes and nucleotides of the invention. Examples of I andII include where X is phenyl substituted with carboxyl, chloro, andother groups (U.S. Pat. Nos. 5,847,162; 6,025,505; 5,654,442; 5,188,934;5,885,778; 6,008,379; 6,020,481; 5,936,087), and where X is hydrogen(Benson, U.S. Pat. No. 6,051,719).

Another class of probe labels include fluorescence quenchers. Theemission spectra of a quencher overlaps with an intermolecularfluorescent dye such that the fluorescence of the fluorescent dye issubstantially diminished, or quenched, by the phenomena of fluorescenceresonance energy transfer “FRET” (Clegg (1992) Meth. Enzymol.,211:353-388). A fluorescent reporter dye and quencher joined on a probein a configuration that permits energy transfer from the fluorophore tothe quencher may result in a reduction of the fluorescence by thefluorescent dye. The reporter is a luminescent compound that can beexcited either by chemical reaction, producing chemiluminescence, or bylight absorption, producing fluorescence. The quencher can interact withthe reporter to alter its light emission, usually resulting in thedecreased emission efficiency of the reporter. The efficiency of thisquenching phenomenon is directly correlated with the distance betweenthe reporter molecule and the quencher molecule (Yaron (1979) AnalyticalBiochemistry, 95:228-35). This self-quenching effect may be diminishedor lost upon hybridization of the probe to its complement or uponnuclease cleavage whereupon the fluorescent reporter and the quencherare separated (FIG. 5).

Particular quenchers include but are not limited to (i) rhodamine dyesselected from the group consisting of tetramethyl-6-carboxyrhodamine(TAMRA), tetrapropano-6-carboxyrhodamine (ROX) (Bergot, U.S. Pat. No.5,366,860):

(ii) aryldiazo compounds, e.g. DABSYL and DABCYL, homologs containingone more additional diazo groups; e.g. Fast Black, (Nardone, U.S. Pat.No. 6,117,986), and substituted compounds where Z is a substituent suchas Cl, F, Br, C₁-C₆ alkyl, C₅-C₁₄ aryl, nitro, cyano, sulfonate, NR₂,—OR, and CO₂H, where each R is independently H, C₁-C₆ alkyl or C₅-C₁₄aryl according to the structures:

(iii) cyanine dyes (Lee, U.S. Pat. No. 6,080,868) such as NTB:

and, (iv) other chromophores e.g. anthraquinone, malachite green,nitrothiazole, and nitroimidazole compounds and the like. The group X isthe covalent attachment site on the primers, probes, and nucleotide5′-triphosphates of the methods of the invention.

Another class of labels serve to effect the separation or immobilizationof labelled amplicons by specific or non-specific capture means, e.g.biotin; 2,4-dinitrophenyl (DNP); and digoxigenin (Andrus, A. “Chemicalmethods for 5′ non-isotopic labelling of PCR probes and primers” (1995)in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp.39-54).

Another class of labels are mobility modifiers, e.g. polyethyleneoxy(PEO) units. The PEO label may be comprised of charged groups, such asphosphodiester to impart charge and increase electrophoretic mobility(velocity). The PEO label may be uncharged and act to retardelectrophoretic or chromatographic mobility. Such modifiers may serve toinfluence or normalize the electrophoretic velocity of amplificationproducts during analysis, e.g. by fluorescent detection, to improveresolution and separation (U.S. Pat. No. 5,470,705)

Another class of probe and primer labels, referred to herein ashybridization-stabilizers, include but are not limited to minor groovebinders, intercalators, polycations, such as poly-lysine and spermine,and cross-linking functional groups. Hybridization-stabilizers mayincrease the stability of base-pairing, i.e. affinity, or the rate ofhybridization (Corey (1995) J. Amer. Chem. Soc. 117:9373-74) of theprimer and target, or probe and target. Hybridization-stabilizers serveto increase the specificity of base-pairing, exemplified by largedifferences in Tm between perfectly complementary oligonucleotide andtarget sequences and where the resulting duplex contains one or moremismatches of Watson/Crick base-pairing (Blackburn, G. and Gait, M. Eds.“DNA and RNA structure” in Nucleic Acids in Chemistry and Biology,2^(nd) Edition, (1996) Oxford University Press, pp. 15-81 and 337-46).Minor groove binders include Hoechst 33258 (Rajur (1997) J. Org. Chem.62:523-29), MGB1 (Gong (1997) Biochem. and Biophys. Res. Comm.240:557-60), and CDPI₁₋₃ (U.S. Pat. No. 5,801,155; WO 96/32496), e.g.CDPI3:

VI.3 Asynchronous Thermal Cycling Protocols

The invention includes novel asynchronous thermal cycling methods forPCR amplification of a target nucleic acid. Targets may be anypolynucleotide capable of primer extension and amplification. Targetnucleic acids include, for example, plasmids, cDNA, amplicons, genomicDNA, restriction digest DNA, and ligation products. Target nucleic acidsmay be polymorphic, including variable repeat sequences and singlenucleotide polymorphisms (SNP). The methods utilize a multi-stageannealing and extension process using primers of disparate Tm values.The PCR amplification reagents include primer extension reagents, suchas a polymerase, nucleotide 5′-triphosphates, and a buffer. Twosignificant advantages may be realized from the methods: (1) targettingss target rather than ds target with probes present in the PCR mixture,and (2) production of an excess or majority of ss amplicon.

The thermal cycling protocols of the invention typically comprise aseries of timed steps at defined temperatures. The series of steps maybe repeated until the PCR process is complete or a desired outcome isachieved, such as detection of certain signals or collection of data.The individual parameters of the steps are selected to optimize theevents in a PCR including: (1) denaturation (thermal melting of a duplexinto single strands); (2) annealing (hybridization of primer to target);and (3) primer extension (incorporation of enzymatically-extendablenucleotides). In some protocols, a probe hybridization step may beincorporated into the cycle. Also, some of the events may be conductedin a single step. For example, probe hybridization and annealing of oneor more of the primers may occur at the same temperature. Annealing andextension of a primer may occur at a single temperature.

The parameters of the steps, e.g. order, duration and temperature, areselected to optimize the outcome and are largely guided by factorsincluding: the Tm of the primers and a probe, if present, the length ofthe amplicon, the amount or purity of target and the detection method.Genomic DNA target sequences of low copy number may necessitate longduration of certain steps or a high number of cycles.

Certain embodiments of the method of the invention includes the step ofdenaturing a double-stranded target nucleic acid at a denaturingtemperature into two strands. FIG. 1 shows a schematic for anasynchronous PCR thermal cycling method according to one embodiment ofthe present invention, including steps of: (i) denaturing doublestranded target, (ii) annealing a first primer, (iii) extension of thefirst primer, (iv) probe hybridization, (v) annealing a second primer,and (vi) extension of the second primer. The temperatures and times aremerely exemplary. In other embodiments, the method may begin with asingle stranded target nucleic acid.

A first, higher-affinity primer is annealed to its complementarysequence of one strand of target at a first annealing temperature(Annealing I in FIG. 1). The higher-affinity primer has a higher Tm thanthe second, lower affinity primer in the reaction vessel. At the firstannealing temperature, the second primer anneals to its complementarysequence on the other strand of target to a lesser extent than the firstprimer anneals to its complementary target sequence because the secondprimer/target duplex does not have sufficient stability at the firstannealing temperature. The first primer is extended by nucleotideincorporation, i.e. addition of nucleotide 5′-triphosphates mediated bypolymerase, at the first annealing temperature, or at an extensiontemperature (Extension I in FIG. 1). The first annealing temperature andthe extension temperature may be a single temperature, at whichannealing and extension of the first primer occur at a commontemperature. At this stage of the method of this embodiment of theinvention, one strand of the target is part of a duplex and the otherstrand is single-stranded.

The temperature may be lowered to a probe hybridization temperature(Hybridization in FIG. 1) at which a detectable probe hybridizes to thesingle-stranded form of the target nucleic acid. The detectable probemay exhibit an increase in fluorescence, e.g. by FRET, uponhybridization or upon cleavage by nuclease activity of the polymerase.

The temperature is then changed to a second annealing temperature(Annealing II in FIG. 1) or kept constant at the probe hybridizationtemperature whereby the second primer anneals to its complementarystrand of the target. The second annealing temperature is lower than thefirst annealing temperature and lower than the extension temperature ofthe first primer. The second primer extends at the second annealingtemperature, or at a higher extension temperature. Extension IItemperature may be the same or different as Extension I temperature. Atthis point in the cycle, a copy of each strand of target has been made.FIG. 1 graphically portrays a cycle of one embodiment of an asynchronousthermal cycling protocol. The temperatures and times of the steps aremerely exemplary.

In the embodiment of the invention illustrated in FIG. 1, the second andsubsequent cycles begin again with denaturing the double strandedtarget, followed by the aforementioned other steps. The cycle may berepeated as many times as desired, but is typically repeated untildetectable signals are evident or stabilize, or until sufficientquantities of amplicon are produced. Typically, 50 cycles are sufficientto detect or produce amplicon. The duration of each step in the cycle issufficient for the completion of the events, i.e. substantially completedenaturation, annealing, extension, and probe hybridization.

An alternative embodiment of an asynchronous thermal cycling protocoldoes not employ a detectable probe or a probe hybridization step. Thisembodiment may be useful when the temporally sequential annealing andextension steps of the first and second primers are conducted in a firststage; Denaturing, Annealing I, Extension I, Annealing II, and ExtensionII, followed by a second stage of a cycle of only the Denaturing,Annealing I and Extension I steps. The first stage may be conducted for2 to 50 cycles, followed by the second stage for 1 to 25 cycles as thelatter portion of the protocol. Omission of the Annealing II andExtension II steps in the second stage allows only, or predominantly,copying of the complement to the first primer. The resulting ampliconwill thus be a preponderance of single-stranded nucleic acid.

In one embodiment, the Tm difference (ΔTm) between the first and secondprimers is large enough such that during the first, higher temperatureannealing and extension steps, only the higher Tm primer undergoesannealing and extension. Typically, annealing temperatures are set 0-10°C. below the Tm of the primer to be annealed and extended. The firstannealing temperature may be any temperature that allows annealing ofthe first primer to target, and that substantially disfavors annealingof the second primer to target. The extension temperature for the firstprimer may be any temperature that allows extension of the first primerto target, and that substantially disfavors annealing of the secondprimer to target for the first primer. The extension temperature of thesecond primer is any temperature that allows extension of the secondprimer to target. The extension temperature of the second primer may bethe same as or different from the second annealing temperature. Duringthe annealing and extension steps of the second, lower Tm primer, mostor substantially all of the target sequence complementary to the first,higher Tm primer has been extended and exists as a duplex, asillustrated in FIG. 1. FIG. 2 shows exemplary Tm ranges for a firstprimer, e.g. Tm=60 to 75° C., and a second primer, e.g. Tm=45 to 55° C.

More than one pair of primers may be present in a PCR reaction conductedby an asynchronous thermal cycling protocol of the invention. More thanone pair of primers may amplify a particular amplicon. When more thanone pair of primers are present in a PCR reaction of the invention, morethan one amplicon may result, i.e. more than one target sequence may beamplified. A particular primer, e.g. a first, higher-melting primer or asecond, lower-melting primer, may form more than one pair of primers andamplify more than one target sequence. For example, a higher-meltingprimer may produce a 100 bp amplicon with one lower-melting primer, anda 200 bp amplicon with a different lower-melting primer. More than probemay be present in a PCR reaction conducted by an asynchronous thermalcycling protocol of the invention. Each probe may have a unique dye andhave a sequence designed to detect a particular target sequencecomplement, e.g. to detect two allelic forms of a gene.

PCR reactions may be conducted in any enclosure or site capable ofthermal cycling. Vessels include tubes, flasks, wells, depressions,frits, porous sites, and addressable locations on surfaces, i.e. arrays.

VI.4 Monitoring Asynchronous PCR with PNA Fret Probes

In one embodiment of the invention, PNA FRET probes labelled with areporter dye and quencher can detect and monitor the real-timeamplification of target polynucleotides by hybridization. PNA probes,complementary to an amplicon sequence internal to the primer sequences,hybridize to ss amplicon after the higher Tm primer has annealed andextended. PNA probes hybridized to complement target are not appreciablycleaved by enzymes, e.g. the exonuclease activity of Taq polymerase,during PCR. When unbound to complement, the reporter dye is quenched.When hybridized to a complementary sequence, the reporter dye andquencher are spatially separated and an increase in fluorescence may bedetected. FIG. 5 shows an exemplary 8-18 nt PNA FRET probe in quenched(separated, unhybridized) and unquenched (hybridized) states. Thefluorescent intensity change may be correlated with hybridization, andthus the presence and quantity of complementary polynucleotide, i.e.amplicon. The PNA FRET probe may be designed to optimize quenching inthe unbound state by incorporating oppositely charged linkers, such ascarboxylate amino acid chains, e.g. glutamic acid and aspartic acid, andammonium amino acid side chains, e.g. lysine. Alternatively, thesequence of the probe may be designed to include non-targetcomplementary sequences at the 5′ and 3′ termini that enforce anintramolecular base-paired conformation, i.e. a hairpin structure, whichbrings the fluorescent dye moiety and the quencher moiety in proximity.

VI.5 Applications of Asynchronous PCR with Real-Time Detection

A step in the real time monitoring of one embodiment of an asynchronousPCR protocol is the hybridization of a detectable probe under highspecificity conditions, i.e. relatively high temperature. Higherspecificity makes single base pair discrimination feasible. The probemay be cleaving, e.g. DNA, or non-cleaving, e.g. PNA or another analog.The probe hybridization and detection step can be conducted at anytemperature and permits the use of very short probes which areintrinsically more specific than corresponding longer ones. As anillustration, FIG. 13 shows a 30-37° C. probe annealing step employedbefore the second primer annealing step. Such a low temperature probeannealing step may be conducted with PNA probes as short as 8 and 9 nt(Example 5, FIG. 8).

A PNA FRET probe binds to DNA target more effectively during theasynchronous thermal cycling PCR protocol than the traditional thermalcycling protocol. FIG. 9 shows the averaged fluorescence changes atcycles 25-30 during each of asynchronous PCR (A-PCR) and traditional(Normal) PCR. At the annealing step at 65° C., the fluorescent intensityincreases only slightly for traditional PCR, indicating less binding ofPNA probes to target, compared to the two-fold signal increase for theA-PCR protocol. The greater increase in signal means more detectionsensitivity, i.e. higher signal/noise at lower copy number of target.

An alternative embodiment of the asynchronous PCR method is to perform afew cycles of a traditional thermal cycling protocol where one of thetwo primers is designed with a high G or C content tail, or “clamp”,such as a 5′ (GC)_(n) or (CC)_(n) where n is 1 to 4. Alternatively, thetail can be a poly G or poly C motif. The GC or CG tail units aredesigned to be non-complementary to any particular target sequence. Thetail serves to increase the Tm of the primer relative to the “untailed”sequence. During the first few cycles, both primers anneal to targetequally well, resulting in relatively synchronous extension during atraditional thermal cycling protocol where the single annealingtemperature is equal or nearly equal to the Tm of the untailed primer.FIG. 14 a shows two cycles of a traditional thermal cycling protocolwith the replication of a GC tail into the amplicon. After severalcycles, the majority of amplicons have incorporated the GC tail at the5′ terminus and the complement to the GC tail at the 3′ terminus. The GCtail of the primer is then complementary to the amplicon and will annealat a higher temperature, at which the untailed primer will not. Afterseveral cycles, e.g. 1 to 5, of the traditional protocol, the thermalcycling protocol can transition to an asynchronous protocol whereby ssamplicon can be targeted by a probe in between the primer annealing andextension steps, or to produce an excess of ss amplicon. Alternatively,the asynchronous protocol may be employed solely. One advantage of theGC tail primer method may be in designing primers or amplicons.

An asynchronous PCR cycle also has utility in a nuclease cleavage assaywith a cleaving DNA FRET probe. One embodiment of the invention providesimprovements to the 5′-exonuclease (TAQMAN®) amplification and detectionprocess (Holland (1991) Proc. Natl. Acad. Sci., 88:7276-80; Livak, U.S.Pat. No. 5,538,848; Gelfand, U.S. Pat. Nos. 5,210,015 and 5,538,848). Apolymerase that conducts primer extension and amplifies thepolynucleotide may also possess a nuclease activity that serves tocleave the phosphodiester bond of a target-annealed probe with anattached “reporter” dye and a “quencher” and where the sequence iscomplementary to the target DNA. Cleavage may release unquenched,labelled fragments for detection. Cleavage of the probe is not necessaryin some assays where detection of a hybridization event is accomplishedby designing a FRET probe in which the spacing between a reporter and aquencher is modulated as a result of the hybridization. (Morrison (1992)in Nonisotopic DNA Probe Techniques, Kricka, ed., Academic Press, Inc.,San Diego, Calif., chapter 13; Heller and Morrison (1985) in RapidDetection and Identification of Infectious Agents, Academic Press, Inc.,San Diego, Calif., pages 245-256). The methods rely on the change influorescence that occurs when suitable fluorescent labels are broughtinto close proximity, variously described in the literature as FRET,fluorescence energy transfer (FET), nonradiative energy transfer,long-range energy transfer, dipole-coupled energy transfer, or Forsterenergy transfer. FRET probes may contain self-complementary, “hairpin”sequences to enforce the “dark” state when unbound to target andincrease specificity in hybridization assays (Tyagi, U.S. Pat. Nos.5,925,517; 6,037,130; 6,103,476; 6,150,097). Examples of systems thatperform the exonuclease assay and other quantitative fluorescent-basedarrays are the ABI PRISM™ 7700, 7200, and 7900HT Sequence DetectionSystems (Applied Biosystems).

VI.6 Applications of Asynchronous PCR with End-Point Detection

The advantages of increased sensitivity and specificity by asynchronousPCR thermal cycling protocols can be realized in assays for humandisease diagnostics, food-borne pathogen detection, and microbialdetection. The resulting amplicons can be detected at the end-point ofPCR by electrophoresis systems such as the ABI PRISM 310, ABI PRISM 377,ABI PRISM 3100, and ABI PRISM 3700 (Applied Biosystems), or onfluorescent plate readers, fluorescence scanners or imaging devices.Amplicons can be detected by PCR with fluorescent dye labelled primersor by intercalator dye staining, e.g. SYBR Green (Molecular Probes,Eugene, Oreg.).

End-point analysis of PCR entails fluorescent dye signal measurementwhen thermal cycling and amplification is complete. Results are reportedin terms of the change in fluorescence, i.e. fluorescence intensityunits, of the fluorescent dye signal from start to finish of the PCRthermal cycling, preferably minus any internal control signals.

Asynchronous PCR thermal cycling protocols of the invention are usefulfor allelic discrimination of target DNA. Probes specific for eachallele can be monitored in a closed-tube, homogeneous PCR assay. Forexample, in a bi-allelic system, two probes can be labelled each with adifferent dye, e.g. FAM and TET, and with sequences specific for eachallelic form (Livak (1995) Nature Genetics 9:341-2; Livak (1999)“Allelic discrimination using fluorogenic probes and the 5′ nucleaseassay” Genetic Analysis: Biomolecular Engineering, Elsevier Press,14:143-49). A mismatch between probe and target greatly reduces theefficiency of probe hybridization, whether the probe is a PNA FRET probeor a nuclease-cleavable DNA FRET probe. Thus, substantial increase inFAM or TET fluorescent signals indicates homozygosity for the FAM- orTET-specific allele. An increase in both signals indicatesheterozygosity.

Asynchronous PCR thermal cycling protocols of the invention may also beuseful for genotyping and gene expression analysis. Genotyping with FRETprobes requires that fluorescence measurements be made after PCR iscompleted (end-point). These types of experiments are convenientlyconducted on the ABI 7200 or 7700 Sequence Detection Systems (AppliedBiosystems). The Systems measure a complete fluorescence spectrum fromabout 500-650 nm directly in PCR reaction tubes. The System softwareautomatically processes the fluorescence data to make genotypedeterminations.

VI.6.a cDNA Library Screening, Homogeneous Sequencing-by-Hybridization(SBH)

Asynchronous PCR may be useful to generate ss cDNA amplicons tocharacterize cDNA libraries. cDNA clones can be grown by normallaboratory procedures on agar plates and inoculated in 96 or 384 wellplates to generate master cultures. DNA purification may be performedusing from 10 to 20 μl cultures on new plates with a correspondingnumber of wells by the boiling method. These procedures can be automated(ABI 6700, Applied Biosystems, Foster City, Calif.). The cDNA insertsmay then be amplified by asynchronous PCR, e.g. in a volume ofapproximately 100 μl in plates. The DNA can be sheared physically into<100 bp fragments if necessary. Each PCR product may then be diluted indistilled, deionized water, e.g. 2×, and aliquotted into 32 identicalmicrotiter plates. The PCR product may then be mixed with one or moreunique PNA FRET probes. Each probe is labelled with unique dyes, e.g.6FAM, TET, HEX, ROX at the amino terminus and a quencher such as NTB,DABCYL at the carboxyl terminus. Fluorescence may then be measured on afluorescence multi-well plate reader, e.g. CytoFluor II (AppliedBiosystems). The resulting normalized and properly scaled fluorescenceintensities of 98 probes to a single clone are indicative ofhybridization and defined as a “hybridization signature” (Drmanac (1993)Science 260:1649-52). The sequence of the hybridizing portion of a cDNAamplicon can be determined by deconvolution of the fluorescenceintensities due to hybridization to a number FRET probes of differentand known sequences (Drmanac (1994) BioTechniques 17:328-9;Milosavljevic (1996) Genome Res. 6:143-141). The normalization of thesignal may be realized by using ratios of the signal for each dye overthe signal from internal control probe targetting a specific plasmidsequence. Hybridization signatures are used to assign the sequencesimilarity between individual clones or cDNA sequences. Clones withsimilar hybridization signatures are grouped into a gene-representingcluster. Clusters are useful to identify specific full-length cDNA ornovel genes based on the difference of cDNA signature profiling amongtissues or treatments.

FIG. 17 shows a schematic of homogeneous SBH using PNA FRET probes. Thesteps of an exemplary method include: (i) cDNA amplified by asynchronousPCR to make ss cDNA amplicons; (ii) ss cDNA amplicons are arrayed; (iii)PNA probes hybridize to each ss cDNA amplicon; (iv) fluorescentdetection gives hybridization signatures. The advantages of the methodinclude: (i) homogeneous conditions; (ii) multiplexed forhigh-throughput applications, i.e. processing many samples in parallel;(iii) rapid hybridization kinetics with short, high Tm PNA probes, and(iv) the cost advantage of shorter probes.

A typical mammalian cell contains between 10,000 to 30,000 differentmRNA sequences. Not all of these mRNA are represented equally in a cDNAlibrary. Low-abundance mRNAs (less than about 10 copies/cell) constituteapproximately 30% of all the mRNAs, and hence there are about 11,000different mRNA that falls into this low-abundance class (Wood (1984)Nature 312:330-7). To achieve a probability of at least 99% of obtainingany rare cDNA clone present in a given cDNA library, up to one millionclones must be screened. FIG. 8 shows the efficient detection withspecificity of sequences with 8 nt and 9 nt PNA FRET probes. A completelibrary of 8 nt PNA FRET probes consists of 4⁸/2=32,000 probes;sufficient to detect the more than one million SNP in the human genomeby cDNA library screening. This library would also be applicable to geneexpression monitoring.

The advantage of the SBH method to cDNA screening is the ability tocharacterize all genes in a cDNA library at once. Assuming one millionclones are needed to characterize a cDNA library, then 2604 plates inthe 384 well format are required for the one million PCR reactions.Asynchronous PCR provides a significant advantage by efficientproduction of single-stranded amplicon ready for hybridization andprecluding amplicon isolation, denaturation and purification. Generationof ss target sequences is often required for efficient hybridization toprobes on an array.

VI.7 Applications for SS DNA Generated by Asynchronous PCR

Asynchronous PCR allows amplification of either + or − strand of DNAtarget, depending on the choice of primer sequence. High Tm primercomplement strand will be formed relative to the low Tm primercomplement strand. Each asynchronous cycle includes two annealing andtwo extension steps. The primers have significantly disparate Tm values,effected largely by primer length. Affinity, as measured by Tm, is alsoaffected by base content (G+C content), sequence, andhybridization-stabilizing labels.

A method to generate a majority of single-stranded DNA amplicon wasdeveloped with a pair of disparate Tm primers. Asynchronous PCR wasconducted for a number of cycles to effect exponential amplification,followed by one or more cycles of thermal cycling with annealing andextension temperatures that only allow hybridization and extension bythe higher melting primer (FIG. 20 b). This serves to linearly amplifyonly one strand of the DNA amplicon, generating an excess, or majority,of ss DNA (FIG. 20 a).

VI.8 Kits

The invention includes kits comprising reagents for amplifying a targetnucleic acid according to the asynchronous PCR methods of the invention.The kits contain a first primer and a second primer. The first primerand second primer have a Tm difference disparate enough such that whilethe first primer anneals and extends to target, the second primer doesnot. Typically, the ΔTm will be about 10 to 30° C. One of the firstprimer or the second primer may be labelled. The label may be afluorescent dye, a mobility modifier, or a hybridization-stabilizingmoiety.

The kits may also contain a detectable probe, a polymerase, andnucleotides. The probe and/or the nucleotides may befluorescent-labelled. The probe may be labelled with a fluorescentmoiety and a quencher moiety. The probe may be DNA, PNA, or a nucleicacid analog.

The kit may contain a set of four different nucleotides, one each thatbears a A, G, C, or T nucleobase. The set may be designed such that thecombination of nucleobases, linkers, and fluorescent dyes yields the setof four nucleotides that result in amplicons that separate underelectrophoresis conditions.

VI.9 EXAMPLES

The invention having been described, the following Examples are offeredby way of illustration, and not limitation. For primer, probe and targetsequences, DNA nucleotides are denoted in upper case letters withmutation sites underlined and in bold. PNA probe sequences are denotedin lower case letters. Unless noted alternatively, the orientation ofDNA sequences is 5′ terminus at the left and 3′ terminus at the right.The orientation of PNA sequences is amino terminus at the left andcarboxyl terminus at the right.

PCR primers and probes in the following examples were designed usingPrimer Express™ (Version 1.0, Applied Biosystems, CA). Thermal melting,Tm, values were estimated for DNA primers and DNA probes by calculationsusing the basic formula:Tm=81.5−16.6(log₁₀[Na⁺]+0.41(% G+C)−(600/N),where N=oligonucleotide length in number of nucleotides (Bolton (1962)Proc. Natl. Acad. Sci., 48:1390; Sambrook, J., Fritsch, E. F., Maniatis,T., Eds. (1989) Molecular Cloning, A Laboratory Manual, Second Edition,Volume 2, pp. 11.46, 9.50-9.51. Refinements to the basic formula may bemade for nearest-neighbor and solvent effects.

Example 1 Melting Temperature Tm Determination of Primers and PNA FRETProbes

Melting temperature (Tm) measurements of PNA FRET probes were performedon either a Lambda 14 spectrophotometer (Perkin-Elmer, Norwalk, Conn.)equipped with a Peltier temperature controller. Temperature ramp rateswere 1° C./min with continuous monitoring at 260 nm. Tm values werecalculated using the maximum values of the first derivative curves ofthe A260 vs. temperature plots using software provided by themanufacturer. Tm determinations were conducted in buffer containing 100mM sodium phosphate and 100 mM sodium chloride. Prior to each Tmmeasurement, each strand of the various DNA templates and PNA probeswere quantified using UV spectroscopy and diluted into the final meltingbuffer at a final concentration of 1 μM. The final optical density rangewas between 0.2 and 0.8 OD (optical density units) at 260 nm. Thesamples were “pre-melted” by heating to 90° C. for 5 min and allowing toslow cool to ambient temperature prior to running the melting profiles.Alternatively, the pre-melts were done on the spectrophotometer byrapidly ramping (5° C./min) up to the high temperature and ramping thetemperature back down to the starting temperature (2-3° C./min) prior torunning the melting profile.

Example 2 PNA FRET Probe Binding Kinetics to ss DNA and ds DNA (FIG. 6)

The kinetics of hybridization of a FRET PNA probe to ss and ds DNA wasmeasured (FIG. 6). When the probe is unbound to target, or below the Tmof the probe in the presence of target, the fluorescent dye and thequencher are in an averaged conformation that allows essentiallycomplete quenching of the fluorescent dye (FIG. 5). When the probe ishybridized to target, the fluorescent dye and quencher are spatiallyseparated and an increase in fluorescence may be measured due to loss ofquenching. Measurement of the fluorescence intensity of a 16 nt PNA FRETprobe (SEQ ID NO:1) gave a baseline of fluorescence. The controlexperiment contains only PNA probe and no target (FIG. 6, top).Quenching is virtually complete throughout the temperature expanse. Amixture of the probe and ds target DNA was held at 95° C. ds DNA wasformed by annealing 68 nt (SEQ ID NO:2) and 74 nt complement (SEQ IDNO:3) to form a 68 bp duplex with 3 nt overhangs. Then the temperaturewas dropped to 60° C. (FIG. 6, middle).

The fluorescence was measured as a function of time in about 5 to 10second intervals over 10 minutes (ABI 7700, Applied Biosystems, FosterCity, Calif.). Fluorescence intensity increased about four times,indicating some hybridization. As the temperature drops to about 60° C.,in the presence of both template strands the binding of the PNA to thecomplementary template strand is out-competed by the other complementaryDNA strand, as seen from the smaller increase in fluorescence (FIG. 6middle). It is also noted that signal slowly drops indicating that thePNA bound is slowly displaced. Finally, a mixture of the probe and sstarget DNA (SEQ ID NO:2) was held at 95° C., then the temperature wasdropped to 60° C. (FIG. 6, bottom). The 16 nt PNA probe binds to ss DNAtarget within a minute, as seen by the eight-fold increase influorescence (FIG. 6 bottom). However, the same probe binds to ds DNAtarget less. Both ss DNA and ds DNA templates ranged from 25, 50, to 100nM. The concentration range was chosen to emulate the PCR stages fromexponential phase to plateau. The results thus show that a probe, e.g.PNA FRET 16 nt hybridizes more rapidly to ss DNA than ds DNA (FIG. 3).The results also demonstrate that probe binding to ds template is bothkinetically and thermodynamically disfavored. PNA 16 nt:FAM-Glu-tgttgccacttcagcc-Lys(dabcyl)-NH2 SEQ ID NO:1 DNA(+ strand)68nt(probe binding region is underlined): 5′TGCGATCCCGCTTGTGATACAGA SEQ IDNO:2 GGCTGAAGTGGCAACA G AGAAGGAAGGAGAAGACGGGGACCAGCC 3′ DNA(− strand)74nt: 5′TTTGGCTGGTCCCCGTCTTCTCCTTCCTTCTCTG SEQ ID NO:3TTGCCACTTCAGCCTCTGTATCACAAGCGGGAT CGCATTT 3′

FAM dabcyl

Example 3 Comparison of Asynchronous, Traditional, and Asymmetric PCRThermal Cycling Protocols (FIGS. 4 a and 4 b)

An asynchronous thermal cycling protocol was directly compared with atraditional thermal cycling protocol. PCR reactions were conducted byindependently varying the following conditions: (i) asynchronous andtraditional (single annealing and single extension steps) thermalcycling protocols; (ii) Tm of the primers; and (iii) concentration ofthe primers. Other conditions were held constant. Target DNA wasamplified with three combinations of forward and reverse primers.

The cycle for asynchronous PCR (A-PCR) is outlined in FIG. 4 a where theprimers are designed so that the Tm values are approximately 15 degreesapart. In the first half of the amplification cycle, the high Tm primeris annealing to the target and then extended fully. Thereafter thetemperature is lowered, e.g. 52° C., and the fluorescence is measured atthis part of the cycle. In this part of the cycle, the low temperatureprimer will bind to the target sequence but may not extend the primer toa substantial extent. The cycle is completed by raising the temperatureand completing the extension of the second primer.

The results from synchronous, traditional, and asymmetric PCR thermalcycling protocols were compared (FIG. 4 b). Two forward primers and tworeverse primers were compared in three of the four possible combinations(66/52; 66/61; 60/61), to create pairs of disparate and nearly equal Tm.Asymmetric PCR was conducted with primers at 200 nM and 20 nMconcentrations. The amplicon and target size was 68 nt. The forwardprimers (Tm=66° C. and 60° C. in each pair of primers) were 5′ labelledwith 6-carboxy fluorescein (FAM) as an electrophoretic mobilitymodifier. The FAM label retards electrophoresis of the amplicons andallow resolution of the strands under denaturing analytical gelconditions. Resolution of labelled (slower migrating) and unlabelled(faster migrating) bands in each lane indicates the presence ofdouble-stranded (FAM labelled, slower migrating, upper band) andsingle-stranded (unlabelled, faster migrating, lower band) ampliconsresulting from PCR under the varied conditions. The electrophoresis wasconducted on 15% polyacrylamide under denaturing conditions (about 55 to60° C. gel temperature during electrophoresis and 7M urea) in thepresence of a SYBR Green™ intercalator (Molecular Probes, Inc., Eugene,Oreg.) to stain and visualize the amplicons.

FIG. 4 b shows the gel electrophoretic analysis of the PCR products uponamplification of the target. The asynchronous PCR with 66° C. and 52° C.Tm primers (3rd lane from the left) gave a 4:1 ratio of upper to lowerbands by densitometry quantitation, and resulted in more amplicon thanthe corresponding traditional PCR with the 66° C. and 52° C. primers. Infact, the asynchronous protocol gave abundant product with all threecombinations of primers whereas the traditional protocol (middle lanes)was only efficient for the nearly equal Tm primer pair (61° C. and 60°C.). The asymmetric thermal cycling protocol (right lanes) wasrelatively inefficient with all three primer combinations. Therefore,FIG. 4 b shows that the asynchronous thermal cycling protocol conductsefficient amplification and allows production of an excess of ssamplicon when disparate Tm primers are employed and the protocol endswith annealing only at the higher temperature. Primers: F1:FAM-TGCGATCCCGCTTGTGATAC SEQ ID NO:4 (Tm = 60° C.) R1:GCTGGTCCCCGTCTTCTCCT SEQ ID NO:5 (Tm = 61° C.) F2:FAM-TGCGATCCCGCTTGTGATACAGA SEQ ID NO:6 (Tm = 66° C.) R2: GGCTGGTCCCCGTC(Tm = 52° C.) SEQ ID NO:7 DNA target, 68 nt:TGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGCAACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:2

PCR primers and double dye-labelled probes were designed using PrimerExpress™ (Version 1.0, Applied Biosystems, CA). Primers were selectedwith varying, disparate Tm and used in three of the four possiblecombinations of the forward and reverse primers. The Tm ranged from 58to 60° C. for primers and 68 to 70° C. for the probes, except shorterPNA FRET probes. Asynchronous PCR primers were designed by adding ordeleting bases of the PCR primers from the 5′ end. At least 15° C.difference in Tm between one (66-75° C.) and the other (50-55° C.) wasexpected.

PCR amplification reactions (50 μl) contained a DNA or RNA targetnucleic acid, 2× Master mix (25 μl) including PCR buffer, dNTPs (dATP,dGTP, dCTP, TTP), and MgCl₂ (Applied Biosystems), AmpliTaq Gold DNApolymerase, forward and reverse primers (200 to 900 nM each), and aprobe (200-250 nM).

Asymmetric PCR:

The 50 μl mixture for asymmetric PCR contained all the reactioncomponents in identical amount as that in the traditional andasynchronous protocols except that the amount of one primer (25-50 pmol)was one twentieth of the other primer (1.25-2.5 pmol). The thermalcycling protocol of the asymmetric PCR was identical to the symmetric,traditional PCR protocol (FIG. 4 b, bottom).

Denaturing PAGE and Image Analysis:

PCR product amplicon (0.5-5 μl) was mixed with a final concentration of1× loading buffer (45 mM Tris base, 45 mM boric acid, 0.4 mM EDTA, 3%Ficoll, 0.02% bromophenol blue, 0.02% xylene cyanol) and denatured at95° C. for 10 to 20 min. The sample was loaded onto a 10-15% denaturingPAGE gel and electrophoresed in 1×TBE (89 mM Tris base, 89 mM boricacid, 2 mM EDTA, pH 8.3) at 100 to 160 V, 70° C. for 25 to 60 min. Theextended product was visualized by staining the gel with 1× SYBR Green(Molecular Probes, Eugene, Oreg.) in a volume of 40 to 120 ml in 1×TBEfor 10 to 30 min. The image was captured by a ChemiImaging 2000 geldocumentation system. The relative amounts of DNA within the bands onthe gel could be compared and ratios calculated by the SpotDenso program(Alpha Innotech Corp., CA).

Example 4 Real-Time Detection of Amplification of Perfect Match andMismatch Targets with Short PNA FRET Probes on the ABI 7700 System(FIGS. 7 a and 7 b)

To demonstrate the achievement of high specificity using an asynchronousPCR method, two different mismatches were installed in the synthetictarget templates; a C_(T) mismatch that is poorly tolerated and a GTmismatch that is generally well accepted, i.e., difficult todiscriminate against. The PNA FRET 16 nt probe (SEQ ID NO:1) readilydiscriminates between the mismatches and the perfect template withseveral cycles between them (FIG. 7 b). Fluorescence is detected duringeach cycle and the logarithmic change in fluorescence (ΔRn) is plottedversus the cycle number. The cycle within the PCR protocol at which thechange in fluorescence (ΔRn) rises above a threshold value is denoted asC_(T). A relatively low C_(T) value indicates efficient detection ofamplicon. The threshold cycle is highly correlated to the amount of copynumber, or amount of target polynucleotide present in the sample. Theperfect match experiment in FIG. 7 b showed probe/target detectionwhereas the mismatch target experiments did not reach the C_(T)threshold. Thus, the 16 nt PNA FRET probe showed single base-pairmismatch specificity. A 14 nt PNA FRET probe (SEQ ID NO:8) complementaryto the same target was prepared and employed with the same cycle andsame primer set as above. The 14 nt PNA FRET probe displayed even betterdiscrimination with amplicons with either mismatched target. Neithermismatch experiment reached the C_(T) threshold and ΔRn is barelyevident even in the late rounds of amplification (FIG. 7 a).

For real-time PCR, the traditional thermal cycling protocol began with 2min at 50° C. and 10 min at 95° C., then proceeded with 40 cycles of 95°C. for 15 sec and 60° C. for 1 min. For real-time asynchronous PCR, eachcycle had two annealing and extension steps including 30 sec at 95 ° C.,30-120 sec at 66-69° C., 30-60 sec at 72° C., 60-120 sec at 52-55° C.,and 60 sec at 72° C. All reactions were performed on the ABI 7700(Applied Biosystems, Foster City, Calif.). Reaction conditions wereprogrammed on a Power Macintosh G3 (Apple Computer, CA) linked directlyto the ABI 7700 Sequence Detector. Analysis of data was also performedon a Macintosh computer with collection and analysis software (AppliedBiosystems). PNA FRET probe 14 nt: FAM-Glu- gt tgc cac ttc agc- SEQ IDNO:8 Lys(dabcyl)-NH₂ PNA FRET probe 16 nt: FAM-Glu-tgt tgc cac ttc agcc- SEQ ID NO:1 Lys(dabcyl)-NH₂ Primers: F2: TGCGATCCCGCTTGTGATACAGA SEQID NO:6 (Tm = 66° C.) R2: GGCTGGTCCCCGTC (Tm = 52° C.) SEQ ID NO:7 DNAtargets: Wild type (perfectly matched)TGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGC A ACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:2 Single-base G-T mismatchedTGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGC G ACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:9 Single-base C-T mismatchedTGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGC C ACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:10

Example 5 Real-Time Detection by Asynchronous PCR with Short PNA FRETProbes (FIG. 8)

Specificity was demonstrated from the sinusoidal correlation between thechange in fluorescence (ΔRn) and the C_(T) threshold (FIG. 8). PCR wasconducted on the ABI 7700 and under the same conditions as in Examples 3and 4. PNA FRET probes: 8 nt: FAM-Glu-tgttgcca-Lys- SEQ ID NO:11Lys(dabcyl)-NH₂ 9 nt: FAM-Glu-tgttgccac-Lys- SEQ ID NO:12Lys(dabcyl)-NH₂ Primers: Forward: GCCCGCCCTGCGATCCCGCTTGTGATAC SEQ IDNO:13 Reverse: GGCTGGTCCCCGTC SEQ ID NO:7 DNA target:TGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGCAACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:2

Example 6 Real-Time Detection by Asynchronous PCR with PNA FRET Probes(FIGS. 10, 11, 12)

A series of three PNA FRET probes, 15-17 nt, and complementary to asynthetic ss 68 nt target DNA (FIG. 21, n=15, 16, 17) were prepared withcarboxyfluorescein as the reporter dye (F) at the N-terminal (equivalentto the 5′-end on DNA) and dabcyl as quencher (Q) on the C-terminal. ThePNA FRET probes were further equipped with a negatively charged glutamicacid moiety between the PNA oligomer and F, and an additional positivelycharged lysine inserted between Q and the PNA oligomer. The oppositelycharged amino acids may tend to enforce proximity of the fluorescent dyeand the quencher and thus a higher degree of quenching when the probe isnot hybridized to a complementary sequence, i.e. target nucleic acid.PCR was conducted on the ABI 7700 and under the same conditions as inExamples 3 and 4.

The PNA FRET probes were used for real-time detection of a synthetic DNAtarget by the asynchronous thermal cycling protocol. The Tm of theprimers differed by 14° C. Target samples were prepared by dilution tocontain 6 different concentrations: 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, and 10⁹starting copies. Each of the three probes (15, 16, 17 nt) were used todetect each target sample concentration by annealing the probe a theprobe annealing step in the protocol and measuring fluorescence,subtracted from background, created by the loss of FRET quenching uponhybridization of probe to target. FIG. 10 shows each probe efficientlydetects amplicon above a threshold fluorescence level as a function ofthe concentration of target. Each amplification is detected by about a20× increase in fluorescence (ΔRn) at the end-point (40 cycles). FIG. 11is a plot of the threshold cycle C_(T) and starting copy number, showinglinear correlation with high correlation coefficiency between the targetsamples and standard controls. By contrast, the same PNA FRET probe andprimers were used to amplify the same target with a traditional thermalcycling protocol (60) cycles. FIG. 12 shows that only the highest copynumber target samples, 10⁸ and 10⁹, gave efficient amplification anddetection. FIG. 12 also reveals a lack of correlation between C_(T) andstarting copy number. None of the traditional protocol amplificationsshowed more than about a 3× increase in fluorescence. PNA FRET probes:15 nt: FAM-Glu-gttgccacttcagcc- SEQ ID NO:14 Lys(dabcyl)-NH₂ (Tm = 70.1°C.) 16 nt: FAM-Glu-tgttgccacttcagcc- SEQ ID NO:1 Lys(dabcyl)-NH₂ (Tm =71.7° C.) 17 nt: FAM-Glu-ctgttgccacttcagcc- SEQ ID NO:15Lys-Lys(dabcyl)-NH₂ (Tm = 72.8° C.) Primers: Forward:TGCGATCCCGCTTGTGATACAGA SEQ ID NO:6 (Tm = 66° C.) Reverse:GGCTGGTCCCCGTC SEQ ID NO:7 (Tm = 52° C.) DNA target (68 bases):TGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGCAACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:2

Example 7 Real-Time Detection of Asynchronous PCR with Three Sets ofPrimers and a PNA FRET Probe on a K-ras Gene Target (FIG. 14 b)

FIG. 14 b shows a real-time detection assay of PCR with three differentpairs of primers and a 16 nt PNA FRET probe on the K-ras gene as thetarget nucleic acid. The assay was conducted on the ABI 7700 with thecycles of FIG. 14 a followed by 40 cycles of an asynchronous thermalcycling protocol. The primer pairs included: (A) equal Tm (52° C.)forward and reverse primers, (B) 5′ (GC)₄ clamp forward primer (Tm 77.5°C.) and reverse primer (Tm 52° C.), and (C) disparate Tm forward andreverse primers (65° C. and 52° C.). It can be seen from the plot of ΔRnduring the course of PCR (FIG. 14 b) that all three primer pairsconducted efficient amplification, with nearly equivalent CT values ofabout 16-17. The GC clamp pair (B) resulted in the largest increase influorescence intensity. The disparate Tm primer pair (C) gave a largerfluorescence intensity increase than the equal Tm primer pair (A), whichforecasts greater sensitivity for low copy number target samples. PCRwas conducted on the ABI 7700 and under the same conditions as inExamples 3 and 4. PNA FRET probe: FAM-O-acg-cca-cca-gct-cca-dabcyl-E SEQID NO:16 Primers: A: Forward: TGCAGAATTCGGCTTAT SEQ ID NO:17 (Tm = 52.5°C.) Reverse: TCGTCCACAAAATGATTC SEQ ID NO:18 (Tm = 52.4° C.) B: Forward:GCGCGCGCTGCAGAATTCGGCTTA SEQ ID NO:19 (Tm = 77.5° C.) Reverse:TCGTCCACAAAATGATTC SEQ ID NO:20 (Tm = 52.4° C.) C: Forward:GACGTTGTAAAACGACGGCCA SEQ ID NO:21 (Tm = 65.3° C.) Reverse:GGATCATATTCGTCCACA SEQ ID NO:22 (Tm = 52.1° C.)

Example 8 Real-Time Detection of the Nuclease Cleavage Assay (FIGS. 15a,b,c)

The asynchronous and traditional thermal cycling protocols were comparedwith a cleaving DNA FRET probe on the ABI 7700 System. Other than theprobe, primers, and target, the PCR amplification reactions containedthe same reagents as Example 2. The target nucleic acid was an ampliconwithin the β-actin gene of genomic DNA.

FIG. 15 a shows the results from detection of PCR using a commercialassay (Applied Biosystems, Foster City, Calif.) with equal Tm primersfor the human β-actin gene in genomic DNA when conducted by traditionalPCR. A series of concentrations of genomic DNA was used, ranging from0.6 pg to 50,000 pg. Eight target samples in this range were employed,at successive 5× difference in concentration. The traditional PCR cyclehas one annealing step and one extension step (FIG. 16, bottom). Primerswith different lengths and disparate Tm values were designed for theassay with the asynchronous thermal cycling protocol (FIG. 16, top).FIG. 15 b shows the results using the disparate Tm primers with theasynchronous thermal cycling protocol and in the otherwise same assayfor the β-actin gene at the eight different concentrations. Bothprotocols were conducted with the same cleavable, DNA FRET probe, SEQ IDNO:23 (FIG. 15 c). Fluorescent signal intensity increased significantlyand the C_(T) values were considerably lower for the asynchronousprotocol (FIG. 15 b) compared to the traditional protocol (FIG. 15 a).The detection limit by the asynchronous protocol allows for single copydetection. In other words, the nuclease cleavage assay is significantlyenhanced by the asynchronous PCR method. The asynchronous PCR method mayalso allow the use of shorter, cleaving DNA FRET probes, i.e. low Tm,under certain conditions. DNA Probe:FAM-ATGCCCTCCCCCATGCCATCCTGCGT-TAMRA SEQ ID NO:23 Primers: traditionalPCR: Forward: ACTGTGCCCATCTACGAGGG SEQ ID NO:24 Reverse:GTGATGACCTGGCAGACGC SEQ ID NO:25 asynchronous PCR: Forward:TGTGCCCATCTACGA SEQ ID NO:26 Reverse: CAGCGGAACCGCTCATTGCCAATGG SEQ IDNO:27

Example 9 End-Point Detection of PCR with 5′-Labelled Primers (FIG. 20a)

To prove that the amplification in A-PCR proceeds in an asynchronousfashion, the forward, higher Tm, primer was 5′ labelled with biotin sothat the two product strands would be well separated during denaturingpolyacrylamide gel electrophoresis. The experimental design is outlinedin FIG. 20 a, bottom. The asynchronous PCR cycle is carried out forfirst 25 cycles then followed by the first half of one cycle wherebyonly the labelled primer hybridizes and extends. The reaction was haltedimmediately by adding 2× loading dye (Novex, San Diego, Calif.) anddenaturing at 95° C. for 20 min. If the amplification is trulyasynchronous then product strands should theoretically be in a 2:1ratio. The ratio was 1:1 when stopped after 25 full cycles, butprogressed to 1:0.67 after the additional one half cycle (FIG. 20 a).This proved that amplification is indeed asynchronous, the highermelting primer preferentially extends, and an excess of single-strandedamplicon is produced. PCR was conducted by 25 cycles of the asynchronousthermal cycling protocol and a final annealing and extension at hightemperature. PCR conditions and analysis employed the conditions ofExample 3. Primers: F1: FAM-TGCGATCCCGCTTGTGATAC SEQ ID NO:4 (Tm = 60°C.) R1: GCTGGTCCCCGTCTTCTCCT (Tm = 61° C.) SEQ ID NO:5 F2:FAM-TGCGATCCCGCTTGTGATACAGA SEQ ID NO:6 (Tm = 66° C.) R2: GGCTGGTCCCCGTC(Tm = 52° C.) SEQ ID NO:7 DNA target:TGCGATCCCGCTTGTGATACAGAGGCTGAAGTGGCAACAG AGAAGGAAGGAGAAGACGGGGACCAGCCSEQ ID NO:2

Example 10 ss DNA Amplification and Labeling by an Asynchronous PCRProtocol (FIG. 22)

The advantage of hybridizing ss amplicons to an array of complementary,solid-phase support bound probes was explored. Two pairs of PCR primerswere designed to compare traditional with asynchronous PCR in generatingamplicons to hybridize to probes spotted on a glass slide array. Theforward primer of each pair had a 5′ Cy5 dye label (Amersham PharmaciaBiotech, Piscataway, N.J.). The reverse primers were unlabelled. The 21nt forward primer and the 20 nt reverse primer of the traditional pairhad approximately equal calculated Tm values. The 25 nt forward primerand the 18 nt reverse primer of the asynchronous pair had a calculatedΔTm of about 15-20° C. The forward primer of the asynchronous pair had a5′ CGGC non-target complementary tail, relative to the forward primer ofthe traditional pair. PCR was conducted to generate a 96 bp ds ampliconby the traditional thermal cycling protocol and a 100 nt ss amplicon bythe asynchronous thermal cycling protocol. Each immobilized probe had a24 nt sequence complementary to each amplicon.

FIG. 22 shows the hybridization of Cy dye 5′-labelled A-PCR (ss DNAmainly) and traditional PCR (ds DNA) products from four differenttargets to a glass slide array. A representative row of the four targetsare enclosed by a rectangle on each array portion for comparison.Signals were normalized by control hybridization. The averaged medianfluorescent signal from labeled A-PCR products (right) was 3- to 4-timeshigher than that from the ds amplicons generated by the traditionalthermal cycling protocol (left). The results suggest that the arrayprobes attached to a glass surface hybridize to ss DNA more effectively.

The target samples contained array probe-specific sequences. PCR wasconducted on the ABI 7700 System. PCR reactions contained 10 mMTris-HCl, pH 8.3, 50 mM KCl, 2-5 mM MgCl₂, 0.01% gelatin, 250 μM eachdNTP, 0.5 to 1 μM forward primer, 0.05 to 0.1 μM reverse primer, 10 μlof 96 nt synthetic target DNA (1:1000 dilution), 1-5 U of AmpliTaq GoldDNA polymerase (Applied Biosystems, Foster City, Calif.) in a totalvolume of 50 μl. The 96 nt synthetic target DNA was prepared bytemplate-dependent ligation of oligonucleotides. Asynchronous PCRincluded two thermal cycling protocols conducted in series. The firstprotocol consisted of an initial 10 min denaturation at 95° C. followedby 15 to 25 cycles of: 95° C. for 15 sec, 65° C. for 60 sec (forwardpriming), 52.5° C. (50-55° C.) for 60 sec (reverse priming), 72° C. for60 sec, and an extra extension of 7 min. The second protocol followedimmediately to produce the single-stranded form of dye-labeled ampliconand consisted of 10 to 80 cycles at 95° C. for 30 sec, 67 (66 to 69)° C.for 90 sec, and 70° C. for 60 sec. Traditional PCR was conducted by theprotocol in Example 4: 2 min at 50° C. and 10 min at 95° C., then 40cycles at 95° C. for 15 sec and 60° C. for 1 min; or 2 min at 50° C. and10 min at 95° C., then 40 cycles at 95° C. for 15 sec, 60° C. for 1 minand 72° C. for 1 min. PCR products were purified in three washes on aMicrocon-100 (Millipore, Medford, Mass.).

Microarray Hybridization, Washing, Data Collection & Analysis

A total of 64 different 24 nt DNA oligonucleotide probes were spotted onglass slides. Eight replicates of each probe were spotted per slide. Thehybridization mixture (20-30 μl/slide) contained 4×SSC (saline-sodiumcitrate), 0.3% SDS (sodium dodecylsulfate), 1 μg/μl, yeast tRNA, 1 μg/μlpoly(A), and 1-2 μl of 50-μl PCR product. The mixture was denatured at95° C. for 2 to 4 min and applied 20-30 μl each to slides. The slide wasplaced inside an array chamber. Following hybridization at 50-55° C. ina waterbath for 16-20 h, the microarrays were washed briefly in 4×SSCand 0.3% SDS at 50-55° C., washed once for 2 min in 1×SSC and 0.3% SDSat room temperature, followed by two washes in 0.06×SSC at roomtemperature for 2 min each. Microarrays were imaged using an Axonscanner, and images were analyzed in GenePix Pro 3.0 software (AxonInstruments, Foster City, Calif.). Traditional primers:Cy5-CCTAGCGTAGTGAGCATCCGT SEQ ID NO:28 ATGCCTCGTGACTGCTACCA SEQ ID NO:29Asynchronous primers: Cy5-CGGCCCTAGCGTAGTGAGCATCCGT SEQ ID NO:30 (Tm =70° C.) ATGCCTCGTGACTGCTAC (Tm = 55° C.) SEQ ID NO:31 DNA target:CCTAGCGTAGTGAGCATCCGTAAGAGCATTCATCGTAGGGGTCTTTGTCCTCTGAGCGTGTACCTGAGAACGGGGATGGTAGCA GTCACGAGGCAT SEQ ID NO:32

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

The invention now having been fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theinvention.

1. A method of nucleic acid amplification comprising the steps of:annealing a first primer to a first strand of a denatured target nucleicacid at a first annealing temperature; extending the first primer withprimer extension reagents at an extension temperature or the firstannealing temperature to generate a double-stranded nucleic acid,wherein the primer extension reagents comprise a polymerase, nucleotide5′-triphosphates, and a buffer; annealing a detectable probe to a secondstrand of the denatured target nucleic acid at a probe hybridizationtemperature; annealing a second primer to the second strand of thedenatured target nucleic acid at a second annealing temperature whereinthe second annealing temperature is lower than the first annealingtemperature and extension temperature; extending the second primer withprimer extension reagents at the extension temperature to generate adouble-stranded nucleic acid; and denaturing the double-stranded targetnucleic acid into a first strand and a second strand at a denaturingtemperature.
 2. The method of claim 1 wherein the detectable probeincludes a fluorescent moiety and a quencher moiety.
 3. The method ofclaim 2 wherein the fluorescent moiety is attached to the 5′ or 3′terminus of the probe and the quencher moiety is attached to the 5′ or3′ terminus of the probe.
 4. The method of claim 1 wherein the probe isdetected prior to extension of the second primer.
 5. The method of claim1 wherein the steps are repeated for 2 to 50 cycles.
 6. The method ofclaim 1 wherein the probe is enzymatically cleaved.
 7. The method ofclaim 1 wherein the probe is not enzymatically cleaved.
 8. The method ofclaim 1 wherein the target nucleic acid is selected from a plasmid, acDNA, an amplicon, genomic DNA, a restriction digest, and a ligationproduct.
 9. The method of claim 1 wherein the target nucleic acidcomprises single nucleotide polymorphisms.
 10. The method of claim 1wherein the first primer and second primer are DNA.
 11. The method ofclaim 1 wherein the first primer or the second primer is a PNA/DNAchimera.
 12. The method of claim 1 wherein the first primer or thesecond primer comprises a covalently attached fluorescent dye.
 13. Themethod of claim 1 wherein the first primer or the second primercomprises a covalently attached mobility-modifier.
 14. The method ofclaim 1 wherein the first primer or the second primer comprises acovalently attached minor groove binder.
 15. The method of claim 1wherein the probe comprises a target-binding sequence and twointramolecularly base-paired sequences.
 16. The method of claim 15wherein the probe forms a hairpin stem and loop structure.
 17. Themethod of claim 15 wherein the intramolecularly base-paired sequencesare at the 5′ terminus and 3′ terminus of the probe.
 18. The method ofclaim 1 wherein the probe comprises one or more nucleotide analogsselected from a nucleobase analog, a 2′-deoxyribose analog, aninternucleotide analog and an optical isomer.
 19. The method of claim 18wherein the nucleobase analog is selected from 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-amino-purine,2,6-diamino-purine, hypoxanthine, pseudouridine, pseudocytidine,pseudoisocytidine, 5-propynyl-cytidine, isocytidine, isoguanine,7-deaza-quanine, 2-thio-pyrimidine, 6-thio-guanine, 4-thio-thymine,4-thio-uracil, O⁶-methyl-guanine, N⁶-methyl-adenine, O⁴-methyl-thymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, andethenoadenine.
 20. The method of claim 18 wherein the nucleotide analogis a 2′-deoxyribose analog that is substituted at the 2′-carbon atomwith Cl, F, —R, —OR, or —NR₂, where each R is independently —H, C₁-C₆alkyl or C₅-C₁₄ aryl.
 21. The method of claim 18 wherein the nucleotideanalog is an LNA.
 22. The method of claim 18 wherein the nucleotideanalog is an L-form optical isomer of 2′-deoxyribose.
 23. The method ofclaim 1 wherein the probe comprises one or more 2-aminoethylglycine(PNA) monomer units.
 24. The method of claim 23 wherein the probe is aPNA/DNA chimera.
 25. The method of claim 1 wherein the probe has thestructure:

wherein: R is a fluorescent moiety; L₁ and L₂ are linkers; B is anucleobase; Q is a quencher moiety; and n is an integer between 5 to 25.26. The method of claim 25 wherein L₁ or L₂ comprise one or more aminoacid units.
 27. The method of claim 26 wherein L₁ and L₂ areindependently selected from aspartic acid, glutamic acid, and lysine.28. The method of claim 27 wherein L₁ is one or more aspartic acid orglutamic acid units, and L₂ is one or more lysine units.
 29. The methodof claim 25 wherein B is selected from uracil, thymine, cytosine,adenine, 7-deazaadenine, guanine, 7-deazaguanosine,7-deaza-8-azaguanine, and 7-deaza-8-azaadenine.
 30. The method of claim2 wherein the fluorescent moiety is a fluorescein dye, a rhodamine dye,or a cyanine dye.
 31. The method of claim 2 wherein the quencher moietyis a rhodamine dye.
 32. The method of claim 2 wherein the quenchermoiety is a nitro-substituted cyanine dye.
 33. The method of claim 2wherein the quencher moiety is selected from the structures:

wherein Z is selected from H, Cl, F, C₁-C₆ alkyl, C₅-C₁₄ aryl, nitro,cyano, sulfonate, NR₂, —OR, and CO₂H, where each R is independently H,C₁-C₆ alkyl or C₅-C₁₄ aryl.
 34. The method of claim 2 wherein a changein fluorescence intensity is detected at the end-point of targetamplification.
 35. The method of claim 2 wherein a change influorescence intensity is monitored in real-time.
 36. The method ofclaim 2 wherein a change in fluorescence intensity is detected as anindication of the presence of the target sequence.
 37. The method ofclaim 1 wherein the first annealing temperature is 10 to 30° C. higherthan the second annealing temperature.
 38. The method of claim 1 whereinthe first annealing temperature is 12 to 16° C. higher than the secondannealing temperature.
 39. The method of claim 1 wherein the firstannealing temperature is 60 to 75° C.
 40. The method of claim 1 whereinthe second annealing temperature is 45 to 55° C.
 41. The method of claim1 wherein the first primer has a (GC)_(n) or a (CG)_(n) sequence at the5′ terminus, where n is 1 to
 4. 42. The method of claim 1 wherein alabel is covalently attached to one or more of the nucleotide5′-triphosphates at the 8-C of a purine nucleobase, the 7-C or 8-C of a7-deazapurine nucleobase, or the 5-position of a pyrimidine nucleobase.43. The method of claim 1 wherein a label is covalently attached to thefirst primer or the second primer at a 5′ terminus, a sugar, aninternucleotide linkage, or a nucleobase.
 44. A method for producingcomplementary polynucleotide strands of a target polynucleotidecomprising: obtaining a mixture comprising first and second targetpolynucleotide strands which are capable of hybridizing with each otherto form a base-paired structure that contains a target sequence, a firstprimer that is complementary to a first region in the first targetpolynucleotide strand, and a second primer that is complementary to asecond region in the second target polynucleotide strand, such that thefirst and second regions flank the target sequence, extending the firstprimer at a first temperature to form a first complex comprising a firstcomplementary strand that is hybridized to the first target strand,under conditions such that the second primer does not substantiallyhybridize to the second region, and extending the second primer at asecond temperature that is lower than the first temperature, to form asecond complex comprising a second complementary strand that ishybridized to the second target strand, wherein before extending thesecond primer, a detectable probe is hybridized to a complementarybinding region in the second target strand, and the hybridized probe isdetected as a measure of second target strand.
 45. A method of nucleicacid amplification comprising the steps of: annealing a first primer toa first strand of a denatured target nucleic acid at a first annealingtemperature; extending the first primer with primer extension reagentsat an extension temperature or the first annealing temperature togenerate a double-stranded nucleic acid, wherein the primer extensionreagents comprise a polymerase, nucleotide 5′-triphosphates, and abuffer; annealing a second primer to a second strand of the denaturedtarget nucleic acid at a second annealing temperature wherein the secondannealing temperature is lower than the first annealing temperature andextension temperature; extending the second primer with primer extensionreagents at the extension temperature to generate a double-strandednucleic acid; and denaturing the double-stranded target into a firststrand and a second strand at a denaturing temperature.
 46. The methodof claim 45 wherein the steps are repeated for 2 to 50 cycles.
 47. Themethod of claim 46 wherein the concentration of the first primer is 2 to10 times higher than the concentration of the second primer.
 48. Themethod of claim 46 wherein the steps of annealing the second primer tothe second strand of the denatured target and extending the secondprimer are omitted in the last 1-25 cycles, whereby a mixture ofsingle-stranded and double-stranded DNA is produced.
 49. The method ofclaim 46 wherein the steps of annealing the second primer to the secondstrand of the denatured target and extending the second primer areomitted in the last 1-10 cycles so as to produce a preponderance of ssDNA.
 50. The method of claim 45 wherein the target is a cDNA.
 51. Themethod of claim 45 wherein the first primer is labelled with afluorescent dye.
 52. The method of claim 45 further comprising the stepof hybridizing the single-stranded and double-stranded DNA productmixture to a plurality of probes immobilized on an array.
 53. The methodof claim 52 wherein the probes are FRET probes.
 54. A method forproducing complementary polynucleotide strands of a targetpolynucleotide comprising: obtaining a mixture comprising a first andsecond target polynucleotides which are capable of hybridizing with eachother to form a base-paired structure that contains a target sequence, afirst primer that is complementary to a first region in the first targetpolynucleotide, and a second primer that is complementary to a secondregion in the second target polynucleotide, such that the first andsecond regions flank the target sequence, extending the first primer ata first temperature to form a first complex comprising a firstcomplementary strand that is hybridized to the first target strand,under conditions such that the second primer does not substantiallyhybridize to the second region, and extending the second primer at asecond temperature that is lower than the first temperature, to form asecond complex comprising a second complementary strand that ishybridized to the second target strand.
 55. The method of claim 54,which further comprises denaturing the first and second complexes afterthe second primer has been extended.
 56. The method of claim 55, whichfurther comprises repeating the first primer extension, second primerextension, and denaturation steps in one or more cycles.
 57. The methodof claim 55, wherein after said denaturation, first primer is extendedat the first temperature to form a mixture comprising the second targetpolynucleotide in single-stranded form and the first complex in duplexform.
 58. A kit for amplifying a target polynucleotide comprising two ormore primers, wherein a first primer and a second primer have a Tmdifference of 10 to 30° C.
 59. The kit of claim 58 wherein a said primeris labelled with a fluorescent dye.
 60. The kit of claim 58 furthercomprising a polymerase.
 61. The kit of claim 58 further comprising adetectable probe.
 62. The kit of claim 61 wherein the detectable probeis DNA and the probe includes a fluorescent moiety and a quenchermoiety.
 63. The kit of claim 61 wherein the detectable probe is PNA andthe probe includes a fluorescent moiety and a quencher moiety.
 64. Thekit of claim 61 wherein the probe comprises a nucleic acid analogselected from a nucleobase analog, a 2′-deoxyribose analog, aninternucleotide analog and an optical isomer.
 65. The kit of claim 58further comprising one or more enzymatically-extendable nucleotides. 66.The kit of claim 65 wherein a nucleotide is labelled with a fluorescentdye.